Modulation of CCR10 signals for treatment of skin and intestinal inflammatory diseases and infection

ABSTRACT

The invention provides compositions and methods for targeting CCR10 and/or the CCR10/ligand axis to modulate the immune response in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/970,451, filed Mar. 26, 2014, the content which is incorporatedby reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos.AI071043 and AR064831, awarded by the National Institutes of Health andunder Hatch Act Project No. PEN04446, awarded by the United StatesDepartment of Agriculture/NIFA. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

As the external surface of a body, skin is under frequent assaults fromenvironmental agents. To maintain its integrity and function, immunecells in the skin are tightly regulated to tolerate harmless antigensbut respond to dangerous assaults. Skin-resident T cells are uniquepopulations of immune cells with memory cell-like properties (Clark, R.A., 2010, J Invest Dermatol 130:362-370). Among them, the resident CD4⁺regulatory T (Treg) cells are critical to maintain the immunehomeostasis of healthy skin (Sather, B. D., et al., 2007, J Exp Med204:1335-1347 and Dudda, J. C., et al., 2008, J Exp Med 205:1559-1565),while the resident memory effector T (Teff) cells could provide fasterimmune responses to infection in the skin than those of the circulation(Gebhardt, T., et al., 2009, Nat Immunol 10:524-530, Jiang, X., et al.,2012, Nature 483:227-231 and Mackay, L. K., et al., 2012, Proc Natl AcadSci USA 109:7037-7042). The antigen encounter in the skin might alsoinduce memory Treg cells to counterbalance the Teff response (Rosenblum,M. D., et al., 2012, Nature 480:538-542). Dysregulation of Treg and Teffcells in the skin is associated with allergy and other skin inflammatorydiseases.

CCR10 and its ligand CCL27 is the most skin-specific chemokine/receptorpair (Zlotnik, A., et al., 2012, Immunity 36:705-716). With CCL27constitutively expressed in healthy skin and further upregulated ininflamed skin in patients of psoriasis and dermatitis and animal models,CCR10/CCL27 have been implicated in migration of skin T cells under bothhealthy and disease conditions (Morales, J., et al., 1999, Proc NatlAcad Sci USA 96:14470-14475, Homey, B., et al., 2000, J Immunol164:3465-3470, Jarmin, D. I., et al., 2000 J Immunol 164:3460-3464 andHomey, B., et al., 2002, Nat Med 8:157-165). In humans, all blood CCR10⁺T cells display memory cell markers, co-express the skin-homing moleculecutaneous lymphocyte antigen (CLA) and respond to chemoattraction ofCCL27, suggesting a role of CCR10/CCL27 in recruitment of memory T cellsinto the skin (Morales, J., et al., 1999, Proc Natl Acad Sci USA96:14470-14475, Homey, B., et al., 2002, Nat Med 8:157-165 and Soler,D., et al., 2003, Blood 101:1677-1682). However, immunohistochemicalstaining in one early study found only scattered CCR10⁺ cells in healthyskin of humans (Homey, B., et al., 2002, Nat Med 8:157-165). Instead,most T cells of inflamed skin of psoriatic and dermatitic patientsexpress CCR10. Since CCL27 was also upregulated in the inflamed skin, itwas suggested that CCR10/CCL27 are involved in migration of T cellsduring the skin inflammation and inhibiting CCR10/CCL27-derived signalscould be used to treat skin inflammatory diseases (Homey, B., et al.,2002, Nat Med 8:157-165). But the flow cytometric analysis of T cellsisolated from the allergen and bacterial chancroid-induced inflamedhuman skin in another study found only a small percentage of CCR10⁺cells, suggesting that CCR10 is unlikely critical for migration of mostT cells during the skin inflammation (Soler, D., et al., 2003, Blood101:1677-1682). A recent study reported that CCR10 is co-expressed onhalf of human blood CCR8⁺ T cells, a population with the skin-homingpotential, but T cells migrating out of the healthy human skin do notexpress CCR10 although non-migrating skin-resident T cells were notassessed (McCully, M. L., et al., 2012, Blood 120:4591-4598). CCR10⁺CD4⁺ T cells of human blood are also enriched with IL-22-producing cellsthat also preferentially express CCR6, another chemokine receptorassociated with skin localization (Duhen, T., et al., 2009, Nat Immunol10:857-863 and Trifari, S., et al., 2009, Nat Immunol 10:864-871). Up tonow, the role of CCR10/CCL27 as homeostatic or inflammatory regulatorsof skin T cells remains unclear.

CCR10 was also expressed on skin-resident innate lymphoid cells (ILC)(Salimi, M., et al., 2013, J Exp Med 210:2939-50). ILC cells are afamily of innate lymphocytes preferentially enriched in barrier tissuessuch as the intestine and skin and involved in the local tissuehomeostasis and inflammation (Spits, H., et al., 2012 Annu Rev Immunol30:647-675). ILCs do not express cell-surface markers associated withother immune cell lineages (lineage marker negative or LIN−) but expressthe common hematopoietic lineage marker CD45 and surface moleculescommonly associated with lymphocytes such as CD90 and CD127 (Spits, H.,et al., 2012 Annu Rev Immunol 30:647-675). Based on their developmentalpathways and functional potentials in analog to the different T helpercell (Th) subsets, ILCs were divided into three major groups (ILC1-3)(Spits, H., et al., 2013, Nat Rev Immunol 13:145-149). ILC1 comprisesnatural killer (NK) cells and other ILCs that produce IFN-γ. ILC2 cellspredominantly express Th2-type cytokines such as IL-5 and IL-15, andILC3 cells produce Th-17/22 type cytokines such as IL-17 and IL-22.Through the production of unique cytokines and direct cell-cellinteraction, ILCs have been reported to act on various other subsets ofimmune cells, such as T cells, mast cells, eosinophils and macrophages,and epithelial cells to regulate homeostasis as well as inflammation inbarrier tissues (Withers, D. R., et al. 2012. J Immunol 189:2094-2098;Hanash, A. M., et al, 2012, Immunity 37:339-350; Dudakov, J. A., et al,2012, Science 336:91-95; Pantelyushin, S., et al., 2012, J Clin Invest122:2252-2256; Roediger, B., et al., 2013, Nat Immunol 14:564-573; Kim,B. S., et al, 2013, Sci Transl Med 5:170ra116; Imai, Y., et al, 2013,Proc Natl Acad Sci USA 110:13921-13926). However, roles of CCR10 inregulating localization and function of ILCs are not clear.

Animal studies also provide complex and sometimes seeminglycontradicting results on functions of CCR10/CCL27. In a mouse model ofthe allergen DNFB (2,4-dinitro-1-fluorobenzene)-induced contacthypersensitive response (CHS), one study found that the antibodyneutralization of CCL27 reduced the skin T cell recruitment andinflammation, suggesting a pivotal role of the CCL27/CCR10 axis in Tcell-mediated skin inflammation (Homey, B., et al., 2002, Nat Med8:157-165). Similarly, in the keratin-14 promoter-driven IL-4 transgenicmouse model of atopic dermatitis, subcutaneous injection of anti-CCL27antibodies reduced inflammation (Chen, L., et al., 2006, Int Immunol18:1233-1242). However, the anti-CCL27 antibody treatment did not affectrecruitment of transferred CD4⁺ T cells into the DNFB-inflamed skin orallergens-induced CHS responses in other studies (Reiss, Y., et al.,2001, J Exp Med 194:1541-1547 and Mirshahpanah, P., et al., 2008, ExpDermatol 17:30-34). Recently, it was reported that CCR10-sufficient and-knockout CD4⁺ T cells migrate similarly to thecognate-antigen-stimulated skin, demonstrating directly that CCR10 isnot critical for the T cell infiltration into inflamed skin (Tubo, N.J., et al., 2011, Am J Pathol 178:2496-2503). Up to now, the role ofCCR10 in regulation of skin immune homeostasis and responses in vivoremains unknown.

CCR10 could be also potentially involved in regulation of immunehomeostasis and response in intestines. In both large and smallintestines, the mucosa-specific ligand for CCR10, CCL28, is highlyexpressed by intestinal epithelial cells (Pan, J., et al, 2000, JImmunol 165:2943-2949; Wang, W., et al, 2000, J Biol Chem275:22313-22323). CCR10 was previously found to express on allIgA-antibody secreting cells (IgA-ASC) and suggested to regulate theirintestinal migration (Kunkel, E. J., et al, 2003, J Clin Invest111:1001-1010). However, anti-CCL28 antibody blockage or CCR10-knockouthad little effects on IgA responses to infections or homeostaticproductions of IgA antibodies in intestines (Feng, N., et al, 2006, JImmunol 176:5749-5759; Morteau, O., et al, 2008, J Immunol181:6309-6315; Hu, S., et al, 2011, Proc Natl Acad Sci USA108:E1035-1044). We found recently that CCR10 is critical in the IgAmemory responses against infections in intestines (Hu, S., et al, 2011,Proc Natl Acad Sci USA 108:E1035-1044), suggesting potentially importantroles of CCR10 in intestinal immune responses. Related with this,expression of the ligand CCL28 was upregulated in inflamed intestines(35)(25). However, up to now, functional importance of CCR10 inintestinal homeostatic regulation and diseases is unknown.

There is a need in the art to better understand the role of CCR10 in theimmune system. In addition, there is a need in the art for thedevelopment of successful therapeutics for the treatment of skin andintestinal diseases. The present invention satisfies the need in the artfor development of new approaches for efficient means to induce animmune response to treat skin and intestinal diseases.

SUMMARY OF THE INVENTION

The invention provides a composition for modulating an immune responsein a mammal. In one embodiment, the composition comprises a modulator ofone or more of CCR10, a ligand of CCR10, and a down-stream effectorprotein in the CCR10/ligand axis.

In one embodiment, the modulator is an inhibitor of one or more ofCCR10, a ligand of CCR10, and a down-stream effector protein in theCCR10/ligand axis, wherein the inhibitor enhances the immune response inthe mammal.

In one embodiment, the inhibitor is selected from the group consistingof a small interfering RNA (siRNA), a microRNA, an antisense nucleicacid, a ribozyme, an expression vector encoding a transdominant negativemutant, an antibody, a peptide and a small molecule.

In one embodiment, the modulator is an activator of one or more ofCCR10, a ligand of CCR10, and a down-stream effector protein in theCCR10/ligand axis, wherein the activator inhibits the immune response inthe mammal.

In one embodiment, the activator is selected from the group consistingof a nucleic acid, a protein, a peptide, a peptidomemetic, a chemicalcompound and a small molecule.

In one embodiment, the composition further comprises a pharmaceuticallyacceptable carrier.

In one embodiment, the composition further comprises an agent selectedfrom the group consisting of an immunostimulatory agent, an antigen, ananti-viral agent, an anti-bacterial agent, an anti-tumor agent, and anycombination thereof.

The invention also provides a method of modulating an immune response ina mammal. In one embodiment, the method comprises administering to themammal in need thereof an effective amount of a composition comprising amodulator of one or more of CCR10, a ligand of CCR10, and a down-streameffector protein in the CCR10/ligand axis.

In one embodiment, the mammal is suffering from a skin disease.

In one embodiment, the skin disease is associated with an infection.

In one embodiment, the skin disease is associated with an inappropriatelevel of immune response that is unable to alleviate the skin disease.

In one embodiment, the mammal is suffering from an autoimmune disease.

In one embodiment, the mammal is suffering psoriasis.

In one embodiment, the mammal is suffering intestinal inflammation.

In one embodiment, the composition is administered in combination with atherapeutic agent to the mammal in need thereof.

In one embodiment, the therapeutic agent is selected from the groupconsisting of an anti-tumor agent, a chemotherapeutic agent, ananti-cell proliferation agent, an anti-tumor vaccine and any combinationthereof.

In one embodiment, the therapeutic agent is administered simultaneously,prior to, or after administration of the modulator.

In one embodiment, the mammal is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1, comprising FIGS. 1A through 1F, is a series of imagesdemonstrating over-reactive innate response to stimulation in skin ofCCR10^(−/−) mice. (FIG. 1A) Ear thickness changes (ΔT) of CCR10^(−/−)and CCR10^(+/−) mice in a DNFB-induced CHS assay. N≥6 mice of eachgenotype in at least two independent experiments (applied in otheranalyses in experiments disclosed herein). (FIG. 1B) Ear thicknesschanges of CCR10^(−/−) and CCR10^(+/−) mice after the one-time topicalapplication of DNFB on the ear. N≥10. *P<0.05; **P<0.005; ***P<0.001 andNS: no significant difference (applied to all figures). (FIG. 1C)qRT-PCR analysis of RNA isolated from treated ears 3 days after theone-time DNFB application for TNF-α, IL-1β and IL-10. N=5 each. “No”indicates untreated skin samples (n=3). The values are relative levelsnormalized on β-actin. (FIG. 1D) Ear thickness changes of CCR10^(−/−)and CCR10^(+/−) mice after the one-time topical application of FITC(0.5%). N=21 for CCR10^(+/−) mice and N=19 for CCR10^(−/−) mice. (FIG.1E) qRT-PCR analysis of RNA isolated from the treated skin 3 days afterthe one-time FITC-application for TNF-α, IL-1β and IL-10 as in (FIG.1C). N=4 each. (FIG. 1F) Ear thickness changes of CCR10^(−/−) andCCR10^(+/−) mice after the one-time topical application of TPA. N≥10 foreach genotype.

FIG. 2, comprising FIGS. 2A through 2K, is a series of imagesdemonstrating imbalanced maintenance and dysregulated functions of Tregand Teff cells in the skin of CCR10^(−/−) mice. (FIG. 2A) Flowcytometric (FACS) analysis of skin lymphocyte preparations ofCCR10^(−/−) and CCR10^(+/−) mice for EGFP⁺ total CD3⁺ T cells (top) andγδT cells (bottom). The number next to each gate is the percentage (%)of the gated cells of total events in the histograph. TheCD3^(high)CCR10^(low) cells are Vγ3⁺ γδT cells. (FIG. 2B) FACS analysisof gated skin EGFP⁺CD3⁺ T cells of CCR10^(−/−) and CCR10^(+/−) mice forCD4⁺ and CD8⁺ subsets. (FIG. 2C) Average percentages of EGFP⁺ CD8⁺ andCD4⁺ cells of skin lymphocyte preparations in CCR10^(+/−) andCCR10^(−/−) mice, calculated by multiplying % of total EGFP⁺ T cells(FIG. 2A) and % of CD4⁺ and CD8⁺ cells of the total EGFP⁺ T cells (FIG.2B). (FIG. 2D) FACS analysis of gated skin EGFP⁺CD4⁺ T cells ofCCR10^(−/−) and CCR10^(+/−) mice for Foxp3⁺ Treg cells. (FIG. 2E)Average percentages of Foxp3⁺CD25⁺ Treg cells of skin CD4⁺ T cells inCCR10^(+/−) and CCR10^(−/−) mice, calculated from the FACS analysis in(FIG. 2D). N≥10. (FIG. 2F) Average numbers of EGFP⁺ CD4⁺Foxp3⁺, CD4⁺ andCD4⁻(CD8⁺) T cells isolated from skin of CCR10^(+/−) vs. CCR10^(−/−)mice. N≥10. (FIG. 2G) FACS analysis of skin CD4⁺ T cells of CCR10^(−/−)and CCR10^(+/−) mice for the IL-10⁺ subset. Average percentages ofIL-10⁺ cells of the EGFP⁺ or EGFP⁻ CD4⁺ cells were shown on the left.N=4 each. (FIG. 2H) FACS analysis of skin CD4⁺ T cells of CCR10^(−/−)and CCR10^(+/−) mice for the IL-17A⁺ subset. The bar graphs show averagepercentages of IL-17A⁺ cells of EGFP⁺ or EGFP⁻ CD4⁺ cells (middle) andtheir MFI for the IL-17A staining (right). N=6 each. (FIG. 2I) Levels ofIL-17A production by purified CCR10^(+/−) or CCR10^(−/−) skinEGFP⁺CD4⁺CD25⁻ T cells stimulated with IL-2 and coatedanti-CD28/anti-TCRβ antibodies in culture. N=4-5. (FIG. 2J) Relativecontribution of transferred CCR10^(−/−) vs. CCR10^(+/−) BM cells to theindicated skin T cell subsets in irradiated WT recipient mice. N=5 each.(FIG. 2K) FACS analysis of skin CD4⁺ T cells of CCR10^(−/−) vs.CCR10^(+/−) BM donor origins in recipient mice for the IL-17A⁺ cells,presented as in (FIG. 2H). N=5 each.

FIG. 3, comprising FIGS. 3A through 3G, is a series of imagesdemonstrating that Treg cells are critical for maintenance of CCR10⁺memory-like resident T cells in the skin. (FIG. 3A) FACS analysis of Tcells of untreated or inflamed skin of CCR10^(+/−) mice for CCR10 (EGFP)expression. The inflamed skin was of mice 1 day after DNFB-induced CHSresponse. The gray lines are of WT cells as negative controls for EGFP.Repeated twice. (FIGS. 3B-3D) FACS analysis for EGFP on skin T cells inRag1^(−/−) mice transferred with CCR10^(+/−) or CCR10^(−/−) EGFP⁻splenic T cells 1 day (FIG. 3B) or 1 month (FIG. 3C, 3D) after theDNFB-induced CHS. Average percentages of EGFP⁺ skin T cells ofCCR10^(+/−) vs. CCR10^(−/−) donors 1 month after the CHS induction areshown in (FIG. 3D). N=10 each. (FIG. 3E) Representative FACS analysisfor molecules associated with memory on skin EGFP⁺ vs. EGFP⁻ T cells ofthe CCR10+/− donor T cell origins in Rag1^(−/−) mice 1 day afterDNFB-induced CHS. N=4. (FIG. 3F) FACS analysis of EGFP expression onTeff (top) and Treg (bottom) cells of the skin of Rag1^(−/−) mice 7-8weeks after they were transferred with EGFP⁻ naïve splenic CD4⁺ Teffcells only (right) or a mixture of EGFP⁻ naïve splenic CD4⁺ Teff andTreg cells (left). N=4. (FIG. 3G) FACS analysis for EGFP on skin T cellsof one-month old CCR10^(+/−) Scurfy and control mice. N=5.

FIG. 4, comprising FIGS. 4A and 4C, is a series of images demonstratingthat CCR10 is critical in localization of CCR10⁺ T cells into thehomeostatic skin. (FIG. 4A-4B) Flow cytometry of gated CD3⁺CD8⁺ T cellsisolated from Ova-treated inflamed ear skin (FIG. 4A) and untreatedtorso skin (FIG. 4B) for the donor-derived OT-I T cells (CD45.1⁻CD45.2⁺)(top) and expression of EGFP of the donor OT-I T cells (bottom). (FIG.4C) Average numbers of EGFP⁺ CCR10^(+/−) and CCR10^(−/−) OT-1 T cellsisolated from the inflamed ear skin and un-affected torso skin based onthe analyses of panel B. N=5-6.

FIG. 5, comprising FIGS. 5A through 5D, is a series of imagesdemonstrating defective resolution of immune memory responses in theskin of CCR10^(−/−) mice. (FIG. 5A) Ear thickness changes of CCR10^(−/−)and CCR10^(+/−) mice in a DNFB-induced memory CHS assay. N≥6. (FIG. 5B)Representative H&E staining of ear sections 3 days after theDNFB-induced memory CHS response (×400). N=4. (FIG. 5C) qRT-PCR analysisof TNF-α, IL-1β and IL-10 transcripts in the treated skin 3 days afterthe DNFB-induced memory CHS. N=4. (FIG. 5D) Ear thickness changes ofRag1^(−/−) mice transferred with EGFP⁻ CCR10^(−/−) and CCR10^(+/−)splenic T cells in the DNFB-induced memory CHS. N≥6.

FIG. 6, comprising FIGS. 6A through 6D, is a series of imagesdemonstrating enhanced immune responses to and accelerated clearance ofLeishmania major infection in skin of CCR10^(−/−) mice. (FIG. 6A) Levelsof Leishmania-specific 16S rDNA in the skin of CCR10^(−/−) andCCR10^(+/−) mice at different time-points after infection, determined byqPCR and normalized on mouse β-actin. N=11, 10 and 4 of each genotypefor 3 days, 1 and 2 months post the infection respectively. (FIG. 6B)qRT-PCR analysis of TNF-α, IL-1β and IL-10 transcripts in the infectedears of CCR10^(−/−) vs. CCR10^(+/−) on Day 3 post the L. majorinfection. N=5 each. (FIG. 6C) Representative ear lesions at infectionsites in CCR10^(−/−) and CCR10^(+/−) mice 1 month after the L. majorinjection. (FIG. 6D) Numbers of total, EGFP⁺ and EGFP⁻ T cells ininfected ears of CCR10^(−/−) and CCR10^(+/−) mice 1 month post theLeishmania infection. N=3 each.

FIG. 7, comprising FIGS. 7A and 7B, is a series of images demonstratingpreferential expression of CCR10 by T cells of the healthy human skin.(FIGS. 7A and 7B) Flow cytometric analysis of gated CD3⁺CD4⁺ T cellsisolated from the healthy skin (FIG. 7A) and blood (FIG. 7B) of humansfor the CCR10 expression. The number in each gate is the averagepercentage of cells in the gate of total events, expressed asmeans±standard errors. N=3 individual samples for each analysis. Theisotype control staining for the CCR10 staining was shown in the leftpanels.

FIG. 8 is an image showing quantitative real-time RT-PCR analysis of RNAisolated from treated ears 3 days after the one-time TPA application forTNF-α, IL-1β and IL-10. “No” indicates untreated skin samples. Thevalues are relative levels normalized on β-actin. N=4 CCR10^(+/−) and 5CCR10^(−/−) mice.

FIG. 9 is an image of a representative flow cytometric analysis of gatedlymphocytes isolated from the skin of CCR10^(+/−) and CCR10^(−/−) micefor CD3⁺TCRβ⁺ T cells and their expression of EGFP (CCR10).

FIG. 10 is an image of a representative flow cytometric analysis ofgated EGFP⁺ CD4⁺ and CD8⁺ T cells isolated from the skin of CCR10^(+/−)and CCR10^(−/−) mice for molecular markers associated with the T cellmemory.

FIG. 11 is an image of a representative FACS analysis of gated splenicCD3+CD8⁺ (top) and CD3+CD4⁺ (bottom) T cells for EGFP⁺ subsets inCCR10^(−/−) and CCR10^(+/−) mice.

FIG. 12, comprising FIGS. 12A and 12B is a series of images showing Tregand Teff cells in the skin of CCR10^(−/−) mice. (FIG. 12A)Representative flow cytometric analysis of gated skin T cell populations(indicated in the figure) for their CCR10^(−/−) vs. CCR10^(+/−) bonemarrow origins in irradiated wild type recipients two months after theBM transfer. CD45.1 and CD45.2 polymorphism was used to distinguish Tcells of different donor and host origins. Total splenic T cells wereanalyzed for their CCR10^(−/−) vs. CCR10^(+/−) bone marrow origins andused as the normalized reference to corresponding skin T cellpopulations. (FIG. 12B) Relative contribution of transferred CCR10^(−/−)vs. CCR10^(+/−) BM cells to the indicated EGFP⁻ skin T cell subsets inirradiated WT recipient mice. N=5 each.

FIG. 13, comprising FIGS. 13A through 13C, is a series of imagesdemonstrating that CCR10 is critical in localization of CCR10⁺ CD4+OT-II T cells into homeostatic skin. (FIG. 13A-13B) Flow cytometry ofgated CD3+CD4+ OT-II cells isolated from Ova-treated inflamed ear skin(FIG. 13A) and untreated torso skin (FIG. 13B) for their expression ofEGFP. (FIG. 13C) Average percentages of CCR10+/− and CCR10−/− OT-II Tcells of the inflamed ear skin and un-inflamed torso skin that expressEGFP (CCR10). N=3 each.

FIG. 14 is an image of a representative flow cytometric analysis of Tcells isolated from L. major-infected ears of CCR10^(−/−) vs.CCR10^(+/−) mice for different T subsets and their expression of EGFP.The mice were analyzed one month after the infection.

FIG. 15 is a schematic illustration of roles of CCR10 in regulation ofskin Treg and Teff cells under homeostatic conditions.

FIG. 16, comprising FIGS. 16A through 16M is a series of imageddemonstrating the identification and characterization of CCR10+ sILCs.FIG. 16A: Flow cytometric analysis of CCR10 (EGFP) expression on ILCsfrom the skin, lamina propria of large intestines (Li-LP) and lungs ofCCR10^(+/EGFP) mice. The contour plot on the left, which is gated onskin CD45⁺ cells, shows the gating strategy for skin_ILCs. Histograms onthe right show the CCR10 (EGFP) expression on ILCs of indicated tissues.Gray areas in histograms are of WT cells as negative controls for EGFP.Skin_ILCs are gated as CD45⁺CD3⁻Lin⁻ cells. Intestinal and lung ILCs aregated as CD45⁺CD3⁻Lin⁻IgA⁻IgM⁻CD138⁻CD19⁻ cells. Representative of >50experiments for the skin, 7 for lungs and 4 for intestines. FIG. 16B&C:Representative flow cytometric analysis of expression of CD127 and CD90(B), and CCR6, CD103 and CD69 (C), on EGFP⁺ and EGFP⁻ skin_ILCs ofCCR10^(+/EGFP) mice. N=3. FIG. 16D: Analysis of expression of CD44 andKi-67 on EGFP⁺ and EGFP⁻ skin_ILCs of CCR10^(+/EGFP) mice. The bar graphon the right compares average percentages of Ki-67⁺ cells among EGFP⁺and EGFP⁻ skin_ILCs. N=3. FIG. 16E: Flow cytometric analysis ofexpression of cytokines IL-17, IL-5, IFN-γ and IL-10 on EGFP⁺ and EGFP⁻skin_ILCs of CCR10^(+/EGFP) mice. Bar graphs on the right showpercentages of IL-17⁺ (N=6) and IL-5⁺ (N=4) cells among EGFP⁺ and EGFP⁻skin_ILCs. N=2 for IFN-γ and IL-10. FIG. 16F: Expression of MHCII onEGFP⁺ and EGFP⁻ skin_ILCs of CCR10^(+/EGFP) mice. N=5. Bar graphs showaverage percentages of MHCII⁺ cells of EGFP⁺ and EGFP⁻ skin_ILCs (left)and median fluorescence intensity (MFI) of their MHCII staining (right).FIG. 16G: Histograms show the expression of different co-stimulatory orco-inhibitory molecules on gated EGFP⁺ skin ILCs of CCR10^(+/EGFP) mice.Gray histograms are of the staining with isotype control antibodies.FIG. 16H: Representative flow cytometric analysis of CCR10 (EGFP)expression on ILCs of skin lymph nodes (sLN), bone marrow (BM), spleenand mesenteric lymph nodes (mLN) of CCR10^(+/EGFP) mice. Gate andanalysis strategies are similar to those in the FIG. 16A. N>30 for sLNand >10 for spleens, 2 for BM and mLN. FIG. 16I&J: Flow cytometricanalysis of expression of CD127 and CD90 (i), CCR6, CD103 and CD69 (j)on EGFP⁺ and EGFP⁻ sLN_ILCs of CCR10^(+/EGFP) mice. N=3. FIG. 16k : Flowcytometric analysis of expression of CD44 and Ki-67 on EGFP⁺ and EGFP⁻sLN_ILCs of CCR10^(+/EGFP) mice. N=5. Bar graph on the right showspercentages of Ki-67⁺ cells among EGFP⁺ and EGFP⁻ sLN_ILCs. FIG. 16L:Flow cytometric analysis of expression of IL-17, IL-5, IFN-γ and IL-10on EGFP⁺ and EGFP⁻ sLN_ILCs of CCR10^(+/EGFP) mice. Bar graphs on theright show percentages of IL-17⁺ (N=7) and IL-5⁺ (N=4) cells among sLNEGFP⁺ and EGFP⁻ ILCs. N=2 for IFN-γ and IL-10. FIG. 16M: Flow cytometricanalysis of MHCII expression on EGFP⁺ and EGFP⁻ sLN_ILCs ofCCR10^(+/EGFP) mice. Bar graph shows percentages of MHCII⁺ cells inEGFP⁺ and EGFP⁻ sLN_ILCs. N=4.

FIG. 17, comprising FIGS. 17A through 17C, is a series of imagescomparing CCR10 (GFP)+ ILCs in sLN (FIG. 17A), skin (FIG. 17B) andspleens (FIG. 17C) of CCR10^(−/−), CCR10^(+/−)CCR6^(−/−),CCR10^(−/−)CCR6^(−/−) and control CCR10^(+/−) littermate mice. Using themice carrying CCR10^(+/−) alleles is to visualize CCR10 expression withthe EGFP reporter. Representatives of flow cytometry of ILCs(CD45+CD3−Lin−) of the different strains were shown on the left. Bargraphs show percentages of EGFP⁺MHCII⁺ cells among ILCs in sLN (N=3) andskin (N=3), as well as percentages of EGFP⁺ cells among ILCs in spleens(N=5). *P<0.05, **P<0.005.

FIG. 18, comprising FIGS. 18A through 18H, is a series of imagesdemonstrating that Treg cells are critical for the generation andmaintenance of CCR10+ sILC cells. FIG. 18A&B: Flow cytometric analysisof expression of CCR10 (EGFP) and MHCII on ILCs from the skin (A) andsLN (B) of CCR10^(+/EGFP)Rag1^(−/−) mice. Plots in the left, gated onCD45⁺ cells, identify CD3⁻Lin⁻ ILCs. Plots in the right are gated onCD45⁺CD3⁻Lin⁻ ILCs. N=3. FIG. 18C: Flow cytometric analysis ofexpression of Ki-67 and CD44 on sLN_ILCs of Rag1^(−/−)CCR10^(+/EGFP)mice. N=3. FIG. 18D: Comparison of MFI of Ki67 and CD44 expressed onEGFP⁻ sLN_ILCs of CCR10^(+/EGFP) (WT) and Rag1^(−/−) CCR10^(+/EGFP)(Rag1^(−/−)) mice. N=3. FIG. 18E: Comparison of percentages of EGFP⁺(left) and EGFP⁺MHCII⁺ (right) cells in skin_ILCs of CCR10^(+/EGFP)(WT), Rag1^(−/−) CCR10^(+/EGFP) (Rag1^(−/−)) mice andRag1^(−/−)CCR10^(+/EGFP) mice transferred with total CD4⁺ cells (+totalCD4) or Treg-depleted CD4⁺ cells (+Foxp3− CD4). For the transfer, equalnumbers of total or Treg-depleted CD4+ T cells (CD3⁺CD4⁺EGFP⁻ orCD3⁺CD4⁺Foxp3− RFP⁻CD62L⁺EGFP⁻) were sorted from spleens ofCCR10^(+/EGFP) (CD45.1⁺CD45.2⁺) mice and injected intoRag1^(−/−)CCR10^(+/EGFP) (CD45.2⁺) mice. Host skin_ILCs were analyzed 6weeks after transfer. N=6 for CCR10^(+/EGFP) mice, 4 forRag1^(−/−)CCR10^(+/EGFP) mice, 5 each for Rag1^(−/−)CCR10^(+/EGFP) micetransferred with total or Treg-depleted CD4⁺ cells. FIG. 18F: Flowcytometric analysis of expression of CCR10 (EGFP) and MHCII on ILCs ofsLN and the skin of Foxp3^(−/−)CCR10^(+/EGFP) (Foxp3^(−/−)) mice. FIG.18G: comparison of percentages of EGFP⁺sLN and skin ILCs betweenCCR10^(+/EGFP) (WT) and Foxp3^(−/−) CCR10^(+/EGFP) (Foxp3^(−/−)) mice.N=4. FIG. 18H: Comparison of percentages of EGFP (CCR10)⁺ cells amongILCs of sLN and the skin of untreated (ctrl, N=6) versuscalcipotriol-treated CCR10^(+/EGFP) mice (treated, N=7).

FIG. 19, comprising FIGS. 19A through 19C, is a series of imagesdemonstrating that CCR10+ sILCs regulate homeostasis of CD4+ cells inthe skin. FIG. 19A: Analysis of donor CD4⁺ T cells for Foxp3⁺ and EGFP⁺subsets in Rag1^(−/−) and CCR10^(EGFP/EGFP)Rag1^(−/−) recipient mice.CD3⁺CD4⁺EGFP⁻ T cells sorted from spleens of CCR10^(+/EGFP)(CD45.1⁺CD45.2⁺) mice were injected into Rag1^(−/−) orCCR10^(EGFP/EGFP)Rag1^(−/−) (CD45.2⁺) mice. Donor CD4⁺ T cells in theskin of recipients were analyzed by flow cytometry 6 weeks aftertransfer. Bar graphs show percentages of Foxp3⁺ and EGFP⁺ subpopulationsamong donor CD4⁺ T cells in the skin of Rag1^(−/−) (N=8) andCCR10^(EGFP/EGFP)Rag1^(−/−) (N=9) recipients. FIG. 19B&C: Analysis oftransferred CD4+ T cells in Rag1^(−/−) and IL2Rγ^(−/−)Rag2^(−/−)recipient mice. The transfer and analysis procedures of the donor CD4+ Tcells in the FIG. 19B are similar as in the FIG. 19A. In addition,percentages of IL-17⁺ subpopulation of donor Foxp3⁻ CD4⁺ T cells in theskin of recipients are determined in the FIG. 19C. N=5 each.

FIG. 20, comprising FIGS. 20A through 20D, is a series of imagesdemonstrating that CCR10^(−/−) mice have increased percentages ofIL-22/IL-17-producing cells in the skin and are prone to developpsoriasis-like skin inflammation symptoms compared to CCR10^(+/−) mice.FIG. 20A: Flow cytometric analysis of skin immune cells of CCR10^(+/−)and CCR10^(−/−) mice for their ability to express IL-22 and IL-17. Gatedon CD45+ cells. A representative of more than 5 experiments is shown.FIG. 20B: Flow cytometric analysis of skin immune cells of agedCCR10^(+/−) and CCR10^(−/−) mice (2 year-old) for their ability toexpress IL-17 and IFN-γ. Gated on CD45+CD3+ T cells. Note that allIL-17-producing T cells are lineage (Lin)-negative (γδ) T cells whilemost IFN-γ producing T cells are Lin+ (αβ) T cells. Representative ofthree experiments. FIG. 20C: Enlarged spleens of aged (2 year-old)CCR10^(−/−) mice compared to those of CCR10^(+/−) mice. Four spleens ofCCR10^(−/−) mice (right) and three of CCR10^(+/−) mice (left) are shown.FIG. 20D: More severe psoriasis-like skin inflammatory symptomes on theskin of CCR10^(−/−) mice compared to the CCR10^(+/−) control 3-6 daysafter treatment of the skin with Imiquimod. Representative of threeexperiments.

FIG. 21, comprising FIGS. 21A through 21E, is a series of imagesdemonstrating that development of spontaneous inflammatory symptoms incolons and impaired recovery from DSS-induced colitis in CCR10^(−/−)mice. FIG. 21A: Length of colons of CCR10^(−/−) and CCR10^(+/−) mice atages of 2-3 and 9 months. One dot represents one mouse. Pictures ofcolons of CCR10^(+/−) and CCR10^(−/−) mice are shown at the right. FIG.21B: Ratios of expression levels of different cytokine transcripts(determined by qRT-PCR) in colons of 9 month-old CCR10^(−/−) over thoseof CCR10^(+/−) mice (1 indicating no difference). N=8. FIG. 21C:Percentages of CCR10^(+/−) and CCR10^(−/−) mice with detectable fecaloccult bleeding 3 days after starting DSS treatment. N=11 forCCR10^(+/−) and N=13 for CCR10^(−/−) mice. FIG. 21D: Survival rates ofCCR10^(+/−) and CCR10^(−/−) mice after starting the DSS treatment. N=10for CCR10^(+/−) and N=9 for CCR10^(−/−) mice. FIG. 21E: Ratios ofexpression levels of indicated cytokine transcripts in colons ofCCR10^(−/−) over those of CCR10^(+/−) mice 7 days after starting DSStreatment. N=5.

FIG. 22, comprising FIGS. 22A through 22E, is a series of imagesdemonstrating that CCR10^(−/−) mice have impaired Treg and Teff cells inthe intestine. FIG. 22A: Analysis of IL-17A, IFNγ, and IL-10 expressionon total CD4⁺ Li_LP T cells of CCR10^(+/−) and CCR10^(−/−) mice. N≥9 forIL-17A, N=4 for IFNγ, and N=15 for IL-10 analyses. FIG. 22B: Flowcytometric analysis of total Li_LP CD4⁺ T cells of CCR10^(+/−) andCCR10^(−/−) mice for EGFP and Foxp3. Gated on CD4+ T cells. Averagepercentages of total and EGFP⁺ Foxp3⁺CD4⁺ Treg cells, and EGFP⁺Foxp3⁻CD4⁺ Teff cells of CCR10^(−/−) and CCR10^(+/−) mice are shown inthree bar graphs on the right. N≥7. FIG. 22C: Percentages of Foxp3⁺ andFoxp3⁻ Li_LP CD4⁺ T cell subsets that express CCR10 (EGFP). N≥7. FIG.22D: Flow cytometric analysis of Li_LP CCR10 (EGFP)+ CD3+CD4+ cells forthe surface IgA signals. For this, the gated EGFP+CD3+ population (ofthe upper right histograph) is analyzed for CD4 and IgA signals (in thelower left histograph). The expression of IgA on the CD3− EGFP+ (mostlyIgA+ cells, upper left histograph) and CD3+ EGFP− (lower righthistograph) populations is also analyzed as controls. FIG. 22E:FlowSight image analysis of cell interaction of intestinal EGFP+ IgA+cells and EGFP− CD3+ T cells. Cellular events that are positive for bothEGFP and CD3 are analyzed using an Amnis FlowSight image cytometer. Notethat EGFP+CD3+ cellular events are composed of two interacting cells(bright field images on the left). In addition, EGFP signals come fromIgA+ cells but not from CD3+ cells of the 2-cell conjugates. Tworepresentatives of more than 30 analyses.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides for compositions and methods for regulating skinand intestinal diseases by modulating CCR10-associated signals or cells.The invention is based on the discovery that targeting CCR10 and/or theCCR10/ligand axis is linked to modulating the immune response in asubject.

Accordingly, the invention provides compositions and methods fortargeting one or more of CCR10, CCR10/ligand axis, and their functionalequivalents for modulating an immune response in a subject. That is, theinvention is based on the novel discovery that CCR10 plays a role inregulating function of the skin and intestinal T and/or ILC cells tomaintain immune homeostasis and prevent overactive immune responses inlocal tissues of skin and intestines. Therefore, by inhibiting one ormore of CCR10, CCR10/ligand axis, and their functional equivalents, animmune response can be enhanced in a subject to treat, for example,diseases and disorders where the immune response is suppressed,including but not limited to skin-specific infections by parasites andcancers. Alternatively, by activating one or more of CCR10, CCR10/ligandaxis, and their functional equivalents, an immune response can bediminished in a subject to treat, for example, diseases and disordersassociated with hyperactive or otherwise undesirable immune response,including but not limited to autoimmune diseases.

In one embodiment, the invention provides compositions and methods formodulating one or more of the level, production, and activity of one ormore of CCR10, CCR10/ligand axis, and their functional equivalents. Inthe context of treating a disease where the immune response issuppressed, the invention provides compositions and methods forinhibiting one or more of the level, production, and activity of one ormore of CCR10, CCR10/ligand axis, and their functional equivalentsthereby enhancing the immune response. In the context of treating adisease associated with an undesirable immune response, the inventionprovides compositions and methods for increasing one or more of thelevel, production, and activity of one or more of CCR10, CCR10/ligandaxis, and their functional equivalents or CCR10+ T and/or ILC cellsthereby diminishing the immune response.

Accordingly, the invention provides activators (e.g., agonists) andinhibitors (e.g., antagonists) of one or more of CCR10, CCR10/ligandaxis, and their functional equivalents. In one embodiment, the activatorof the invention includes but is not limited to a small molecule, achemical compound, a protein, a peptide, a peptidomemetic, a nucleicacid, and the like. In one embodiment, the inhibitor of the inventionincludes but is not limited to an antibody or a fragment thereof, apeptide, a nucleic acid, small molecule, a chemical compound, and thelike.

In one embodiment, the present invention comprises a method forincreasing one or more of the level, production, and activity of one ormore of CCR10, CCR10/ligand axis, and their functional equivalentscomprising administering to a subject an effective amount of acomposition comprising an activator of one or more of CCR10,CCR10/ligand axis, and their functional equivalents. In an embodiment ofthe present invention, the composition increases the transcription ofone or more of CCR10, CCR10/ligand axis, and their functionalequivalents or translation of one or more of CCR10, CCR10/ligand axis,and their functional equivalents. In another embodiment of the presentinvention, the composition increases the activity of one or more ofCCR10, CCR10/ligand axis, and their functional equivalents.

An aspect of the present invention comprises a method for decreasing oneor more of the level, production, and activity of one or more of CCR10,CCR10/ligand axis, and their functional equivalents comprisingadministering to a subject an effective amount of a compositioncomprising an inhibitor of one or more of CCR10, CCR10/ligand axis, andtheir functional equivalents. In an embodiment of the present invention,the composition decreases the transcription of one or more of CCR10,CCR10/ligand axis, and their functional equivalents or translation ofone or more of CCR10, CCR10/ligand axis, and their functionalequivalents. In another embodiment of the present invention, thecomposition inhibits the activity of one or more of CCR10, CCR10/ligandaxis, and their functional equivalents.

Another aspect of the present invention comprises a pharmaceuticalcomposition comprising a modulator (e.g., activator or inhibitor) of oneor more of CCR10, CCR10/ligand axis, and their functional equivalents.In one embodiment, the composition of the invention can be used incombination with another therapeutic agent.

In one embodiment, the invention provides compositions and methods fortreating skin diseases associated with an ineffective immune response.In another embodiment, treatment of the skin disease is accomplished byinducing an enhanced immune response.

In one embodiment, the present invention provides compositions andmethods for enhancing an immune response in a subject by modulating oneor more of CCR10, CCR10/ligand axis, and their functional equivalents.The present invention includes the use of the compounds of the inventionin combination with vaccines and other therapies in which it isdesirable to enhance the immune response by inhibiting one or more ofCCR10, CCR10/ligand axis, and their functional equivalents.

In addition, the present invention also provides compositions andmethods for breaking self-tolerance in tumor vaccination. Therefore thepresent invention includes a therapeutic benefit of enhancing the immuneresponse in a subject by interfering with one or more of CCR10,CCR10/ligand axis, and their functional equivalents.

In one embodiment, the invention provides compositions and methods fortreating an autoimmune disease. In one embodiment, the autoimmunedisease is psoriasis. In another embodiment, the autoimmune disease isautoimmune intestinal inflammation. Therefore the present inventionincludes a therapeutic benefit of inhibiting the immune response in asubject by activating one or more of CCR10, CCR10/ligand axis, and theirfunctional equivalents.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)₂, as well as single chain antibodies and humanizedantibodies (Harlow et al., 2001, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426).

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFvantibodies, and multispecific antibodies formed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. κ and λ light chains refer tothe two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

“Antisense” refers particularly to the nucleic acid sequence of thenon-coding strand of a double stranded DNA molecule encoding apolypeptide, or to a sequence which is substantially homologous to thenon-coding strand. As defined herein, an antisense sequence iscomplementary to the sequence of a double stranded DNA molecule encodinga polypeptide. It is not necessary that the antisense sequence becomplementary solely to the coding portion of the coding strand of theDNA molecule. The antisense sequence may be complementary to regulatorysequences specified on the coding strand of a DNA molecule encoding apolypeptide, which regulatory sequences control expression of the codingsequences.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

The term “B-cell” as used herein is defined as a cell derived from thebone marrow and/or spleen. B cells can develop into plasma cells whichproduce antibodies.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike.

The term “CCR10-mediated disorder” refers to disease states and/orsymptoms associated with CCR10-mediated infections, cancers or tumors.In general, the term “CCR10-mediated disorder” refers to any disorder,the onset, progression or the persistence of the symptoms of whichrequires the participation of CCR10. Exemplary CCR10-mediated disordersinclude, but are not limited to, infection and cancer.

The term “CCR10/ligand axis” refers to the signaling pathway associatedwith the chemokine/receptor pair of skin-associated chemokine CCL27(also called CTACK, ALP and ESkine) and its receptor CCR10 (GPR-2).

As used herein, an “effector cell” refers to a cell which mediates animmune response against an antigen. An example of an effector cellincludes, but is not limited to a T cell and a B cell.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

The term “effector cell” refers to a cell which mediates an immuneresponse against an antigen. An example of an effector cell includes,but is not limited to, a T cell or a B cell.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

The term “expression vector” as used herein refers to a vectorcontaining a nucleic acid sequence coding for at least part of a geneproduct capable of being transcribed. In some cases, RNA molecules arethen translated into a protein, polypeptide, or peptide. In other cases,these sequences are not translated, for example, in the production ofantisense molecules, siRNA, ribozymes, and the like. Expression vectorscan contain a variety of control sequences, which refer to nucleic acidsequences necessary for the transcription and possibly translation of anoperatively linked coding sequence in a particular host organism. Inaddition to control sequences that govern transcription and translation,vectors and expression vectors may contain nucleic acid sequences thatserve other functions as well.

The term “heterologous” as used herein is defined as DNA or RNAsequences or proteins that are derived from the different species.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 5′-ATTGCC-3′ and 5′-TATGGC-3′ share 50%homology.

As used herein, “homology” is used synonymously with “identity.”

The term “immunoglobulin” or “Ig”, as used herein is defined as a classof proteins, which function as antibodies. The five members included inthis class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is theprimary antibody that is present in body secretions, such as saliva,tears, breast milk, gastrointestinal secretions and mucus secretions ofthe respiratory and genitourinary tracts. IgG is the most commoncirculating antibody. IgM is the main immunoglobulin produced in theprimary immune response in most mammals. It is the most efficientimmunoglobulin in agglutination, complement fixation, and other antibodyresponses, and is important in defense against bacteria and viruses. IgDis the immunoglobulin that has no known antibody function, but may serveas an antigen receptor. IgE is the immunoglobulin that mediatesimmediate hypersensitivity by causing release of mediators from mastcells and basophils upon exposure to allergen.

As used herein, the term “immune response” includes, but is not limitedto, T cell-mediated and/or B cell-mediated immune responses that areinfluenced by modulation of T cell costimulation. Exemplary immuneresponses include B cell responses (e.g., antibody production) T cellresponses (e.g., cytokine production, and cellular cytotoxicity) andactivation of cytokine responsive cells, e.g., macrophages.

As used herein, the term “immune cell” is intended to include, but isnot limited to, a cell that is of hematopoietic origin and that plays arole in the immune response. Immune cells include, but are not limitedto, lymphocytes, such as B cells and T cells; natural killer cells; andmyeloid cells, such as monocytes, macrophages, eosinophils, mast cells,basophils, and granulocytes.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, i.e., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, i.e., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, i.e., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (i.e.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

As used herein, the term “modulate” is meant to refer to any change inbiological state, i.e. increasing, decreasing, and the like. Forexample, the term “modulate” refers to the ability to regulatepositively or negatively the expression or activity of CCR10, includingbut not limited to transcription of CCR10 mRNA, stability of CCR10 mRNA,translation of CCR10 mRNA, stability of CCR10 polypeptide, CCR10post-translational modifications, or any combination thereof. Further,the term modulate can be used to refer to an increase, decrease,masking, altering, overriding or restoring of activity, including butnot limited to, CCR10 activity associated with immunogenicity of a cell.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

The term “polypeptide” as used herein is defined as a chain of aminoacid residues, usually having a defined sequence. As used herein theterm polypeptide is mutually inclusive of the terms “peptide” and“protein.”

As used herein, a “peptidomimetic” is a compound containing non-peptidicstructural elements that is capable of mimicking the biological actionof a parent peptide. A peptidomimetic may or may not comprise peptidebonds.

“Proliferation” is used herein to refer to the reproduction ormultiplication of similar forms of entities, for example proliferationof a cell. That is, proliferation encompasses production of a greaternumber of cells, and can be measured by, among other things, simplycounting the numbers of cells, measuring incorporation of ³H-thymidineinto the cell, and the like.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

The term “RNA” as used herein is defined as ribonucleic acid.

The term “self-antigen” as used herein is defined as an antigen that isexpressed by a host cell or tissue. Self-antigens may be tumor antigens,but in certain embodiments, are expressed in both normal and tumorcells. A skilled artisan would readily understand that a self-antigenmay be overexpressed in a cell.

The terms “subject,” “patient,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are culture in vitro. In other embodiments, the cells are notcultured in vitro.

The term “T-cell” as used herein is defined as a thymus-derived cellthat participates in a variety of cell-mediated immune reactions.

The term “T-helper” as used herein with reference to cells indicates asub-group of lymphocytes (a type of white blood cell or leukocyte)including different cell types identifiable by a skilled person. Inparticular, T-helper cell according to the present disclosure includeeffector T_(h) cells (such as Th1, Th2 and Th17). These Th cells secretecytokines, proteins or peptides that stimulate or interact with otherleukocytes.

The term “regulatory T cell” or “Treg cell” refers to a naturallyoccurring subtype of T cell that can inhibit T-cell immune responses toan antigen. Treg cells represent a distinct T-cell lineage that has akey role in an individual's tolerance of self-antigens and theprevention of autoimmune disease and inappropriate immune responses.When activated, they are anergic and suppress the proliferation andcytokine production of conventional T cells. Like all T cells, Tregcells require T cell receptor activation and costimulation to becomefully active.

“Therapeutically effective amount” is an amount of a compound of theinvention, that when administered to a patient, ameliorates a symptom ofthe disease. The amount of a compound of the invention which constitutesa “therapeutically effective amount” will vary depending on thecompound, the disease state and its severity, the age of the patient tobe treated, and the like. The therapeutically effective amount can bedetermined routinely by one of ordinary skill in the art having regardto his own knowledge and to this disclosure.

“Patient” for the purposes of the present invention includes humans andother animals, particularly mammals, and other organisms. Thus themethods are applicable to both human therapy and veterinaryapplications. In a preferred embodiment the patient is a mammal, and ina most preferred embodiment the patient is human.

The terms “treat,” “treating,” and “treatment,” refer to therapeutic orpreventative measures described herein. The methods of “treatment”employ administration to a subject, in need of such treatment, acomposition of the present invention, for example, a subject having adisorder mediated by CCR10 or other oncoprotein or a subject whoultimately may acquire such a disorder, in order to prevent, cure,delay, reduce the severity of, or ameliorate one or more symptoms of thedisorder or recurring disorder, or in order to prolong the survival of asubject beyond that expected in the absence of such treatment.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

The term “vaccine” as used herein is defined as a material used toprovoke an immune response after administration of the material to amammal.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The disclosure presented herein demonstrates a new role for CCR10 in themaintenance and function of the resident regulatory T (Treg) vs.effector T (Teff) cells. For example, the invention is based on thesurprising discovery that CCR10 plays a role in a positive feedbackcircuit of immune homeostatic regulation in the skin where CCR10maintains balanced presence and function of resident Treg vs. Teffcells, which in turn establishes a homeostatic environment important formaintenance of the CCR10+ resident T cells. Accordingly, the inventionincludes compositions and methods for targeting CCR10 for drug therapy.In some instances, inhibiting CCR10 is useful in increasing the immuneresponse and therefore allowing the immune system to respond to thetarget. In some instances, activating CCR10 is useful in inhibiting theimmune response.

The present invention provides compounds and methods for inhibitingCCR10, such as the CCR10/ligand axis and methods of treating diseasesmediated by activity of CCR10 and functionally equivalents thereof usingthe compounds of the invention. The invention also provides compoundsand methods of inhibiting downstream targets of CCR10 and its functionalequivalents. Diseases mediated by CCR10 and functionally equivalentsthereof include, but are not limited to, diseases characterized in partby abnormalities in optimal immune reaction, infection, cancer, and thelike.

The present invention provides compounds and methods for activatingCCR10, such as the CCR10/ligand axis and methods of treating diseasesmediated by activity of CCR10 and functionally equivalents thereof usingthe compounds of the invention. The invention also provides compoundsand methods of activating downstream targets of CCR10 and its functionalequivalents. Diseases mediated by CCR10 and functionally equivalentsthereof include, but are not limited to, autoimmune disease and diseasesassociated with hyperactive immune response.

Compositions

In one embodiment, the invention provides a modulator (e.g., aninhibitor or activator) of one or more of CCR10, a ligand of CCR10, anda down-stream effector protein in the CCR10/ligand axis. In variousembodiments, the present invention includes compositions for modulatingthe level or activity of one or more of CCR10, a ligand of CCR10, and adown-stream effector protein in the CCR10/ligand axis in a subject, acell, a tissue, or an organ in need thereof. In various embodiments, thecompositions of the invention modulates the amount of polypeptide of oneor more of CCR10, a ligand of CCR10, and a down-stream effector proteinin the CCR10/ligand axis, the amount of mRNA of one or more of CCR10, aligand of CCR10, and a down-stream effector protein in the CCR10/ligandaxis, the amount of activity of one or more of CCR10, a ligand of CCR10,and a down-stream effector protein in the CCR10/ligand axis, or acombination thereof.

In one embodiment, an inhibitor of the invention is useful for treatingtreat, for example, diseases and disorders where the immune response issuppressed, including but not limited to skin-specific infections byparasites and cancers. In one embodiment, an activator of the inventionis useful for treating for example, diseases and disorders associatedwith hyperactive or otherwise undesirable immune response, including butnot limited to autoimmune diseases.

Inhibitors

As described elsewhere herein, the invention is based on the discoverythat inhibition of CCR10 enhances the immune response in a subject. Thisobservation is the first of its kind by providing a direct link betweenfunction of CCR10 in a positive feedback circuit of immune homeostaticregulation in the skin where CCR10 maintains balanced presence andfunction of resident Treg vs. Teff cells, which in turn help toestablish a homeostatic environment important for maintenance of theCCR10+ resident T cells. As such the invention provides compositions andmethods for therapeutic inhibition of CCR10 in a subject. The resultspresented herein also provide for combining any immunotherapy protocolsin diseases such as skin infection and cancer with inhibitors of theinvention that targets CCR10.

The present invention relates to the discovery that inhibition of one ormore of CCR10, CCR10/ligand axis, and their functional equivalentsprovides a therapeutic benefit. Thus, the invention comprisescompositions and methods for inhibiting any of these components in cellor animal thereby enhancing an immune response in the animal.

Based on the disclosure herein, the present invention includes a genericconcept for inhibiting CCR10 or any component of the signal transductionpathway associated with CCR10 or a functional equivalent thereof.

In one embodiment, the invention comprises a composition for enhancingan immune response. The composition comprises an inhibitor of any one ormore of CCR10, CCR10/ligand axis, and their functional equivalents. Inone embodiment, composition comprising an inhibitor wherein theinhibitor includes but is not limited to a small interfering RNA(siRNA), a microRNA, an antisense nucleic acid, a ribozyme, anexpression vector encoding a transdominant negative mutant, anintracellular antibody, a peptide and a small molecule.

By way of a non-limiting example, siRNA is a type of molecule that caninhibit CCR10. An siRNA polynucleotide is an RNA nucleic acid moleculethat interferes with RNA activity that is generally considered to occurvia a post-transcriptional gene silencing mechanism. An siRNApolynucleotide preferably comprises a double-stranded RNA (dsRNA) but isnot intended to be so limited and may comprise a single-stranded RNA(see, e.g., Martinez et al., 2002 Cell 110:563-74). The siRNApolynucleotide included in the invention may comprise other naturallyoccurring, recombinant, or synthetic single-stranded or double-strandedpolymers of nucleotides (ribonucleotides or deoxyribonucleotides or acombination of both) and/or nucleotide analogues as provided herein(e.g., an oligonucleotide or polynucleotide or the like, typically in 5′to 3′ phosphodiester linkage). Accordingly it will be appreciated thatcertain exemplary sequences disclosed herein as DNA sequences capable ofdirecting the transcription of the siRNA polynucleotides are alsointended to describe the corresponding RNA sequences and theircomplements, given the well-established principles of complementarynucleotide base-pairing.

Preferred siRNA polynucleotides comprise double-stranded polynucleotidesof about 18-30 nucleotide base pairs, preferably about 18, about 19,about 20, about 21, about 22, about 23, about 24, about 25, about 26, orabout 27 base pairs, and in other preferred embodiments about 19, about20, about 21, about 22 or about 23 base pairs, or about 27 base pairs,whereby the use of “about” indicates that in certain embodiments andunder certain conditions the processive cleavage steps that may giverise to functional siRNA polynucleotides that are capable of interferingwith expression of a selected polypeptide may not be absolutelyefficient. Hence, siRNA polynucleotides, may include one or more siRNApolynucleotide molecules that may differ (e.g., by nucleotide insertionor deletion) in length by one, two, three, four or more base pairs as aconsequence of the variability in processing, in biosynthesis, or inartificial synthesis of the siRNA. The siRNA polynucleotide of thepresent invention may also comprise a polynucleotide sequence thatexhibits variability by differing (e.g., by nucleotide substitution,including transition or transversion) at one, two, three or fournucleotides from a particular sequence. These differences can occur atany of the nucleotide positions of a particular siRNA polynucleotidesequence, depending on the length of the molecule, whether situated in asense or in an antisense strand of the double-stranded polynucleotide.The nucleotide difference may be found on one strand of adouble-stranded polynucleotide, where the complementary nucleotide withwhich the substitute nucleotide would typically form hydrogen bond basepairing, may not necessarily be correspondingly substituted. Inpreferred embodiments, the siRNA polynucleotides are homogeneous withrespect to a specific nucleotide sequence.

Based on the present disclosure, it should be appreciated that thesiRNAs of the present invention may effect silencing of the targetpolypeptide expression to different degrees. The siRNAs thus must firstbe tested for their effectiveness. Selection of siRNAs is made therefrombased on the ability of a given siRNA to interfere with or modulate theexpression of the target polypeptide. Accordingly, identification ofspecific siRNA polynucleotide sequences that are capable of interferingwith expression of a desired target polypeptide requires production andtesting of each siRNA. The methods for testing each siRNA and selectionof suitable siRNAs for use in the present invention are fully set forthherein the Examples. Since not all siRNAs that interfere with proteinexpression will have a physiologically important effect, the presentdisclosure also sets forth various physiologically relevant assays fordetermining whether the levels of interference with target proteinexpression using the siRNAs of the invention have clinically relevantsignificance.

One skilled in the art will readily appreciate that as a result of thedegeneracy of the genetic code, many different nucleotide sequences mayencode the same polypeptide. That is, an amino acid may be encoded byone of several different codons, and a person skilled in the art canreadily determine that while one particular nucleotide sequence maydiffer from another, the polynucleotides may in fact encode polypeptideswith identical amino acid sequences. As such, polynucleotides that varydue to differences in codon usage are specifically contemplated by thepresent invention.

One skilled in the art will appreciate, based on the disclosure providedherein, that one way to decrease the mRNA and/or protein levels of oneor more of CCR10, CCR10/ligand axis, and their functional equivalents isby reducing or inhibiting expression of the nucleic acid encoding CCR10or any component of the signal transduction pathway associated withCCR10 or a functional equivalent thereof. Thus, the protein level ofCCR10 or any component of the signal transduction pathway associatedwith CCR10 or a functional equivalent thereof in a cell can also bedecreased using a molecule or compound that inhibits or reduces geneexpression such as, for example, an antisense molecule or a ribozyme.

In a preferred embodiment, the modulating sequence is an antisensenucleic acid sequence which is expressed by a plasmid vector. Theantisense expressing vector is used to transfect a mammalian cell or themammal itself, thereby causing reduced endogenous expression of adesired regulator in the cell. However, the invention should not beconstrued to be limited to inhibiting expression of a regulator bytransfection of cells with antisense molecules. Rather, the inventionencompasses other methods known in the art for inhibiting expression oractivity of a protein in the cell including, but not limited to, the useof a ribozyme, the expression of a non-functional regulator (i.e.transdominant negative mutant) and use of an intracellular antibody.

Antisense molecules and their use for inhibiting gene expression arewell known in the art (see, e.g., Cohen, 1989, In:Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRCPress). Antisense nucleic acids are DNA or RNA molecules that arecomplementary, as that term is defined elsewhere herein, to at least aportion of a specific mRNA molecule (Weintraub, 1990, ScientificAmerican 262:40). In the cell, antisense nucleic acids hybridize to thecorresponding mRNA, forming a double-stranded molecule therebyinhibiting the translation of genes.

The use of antisense methods to inhibit the translation of genes isknown in the art, and is described, for example, in Marcus-Sakura (1988,Anal. Biochem. 172:289). Such antisense molecules may be provided to thecell via genetic expression using DNA encoding the antisense molecule astaught by Inoue, 1993, U.S. Pat. No. 5,190,931.

Alternatively, antisense molecules of the invention may be madesynthetically and then provided to the cell. Antisense oligomers ofbetween about 10 to about 30, and more preferably about 15 nucleotides,are preferred, since they are easily synthesized and introduced into atarget cell. Synthetic antisense molecules contemplated by the inventioninclude oligonucleotide derivatives known in the art which have improvedbiological activity compared to unmodified oligonucleotides (see U.S.Pat. No. 5,023,243).

Ribozymes and their use for inhibiting gene expression are also wellknown in the art (see, e.g., Cech et al., 1992, J. Biol. Chem.267:17479-17482; Hampel et al., 1989, Biochemistry 28:4929-4933;Eckstein et al., International Publication No. WO 92/07065; Altman etal., U.S. Pat. No. 5,168,053). Ribozymes are RNA molecules possessingthe ability to specifically cleave other single-stranded RNA in a manneranalogous to DNA restriction endonucleases. Through the modification ofnucleotide sequences encoding these RNAs, molecules can be engineered torecognize specific nucleotide sequences in an RNA molecule and cleave it(Cech, 1988, J. Amer. Med. Assn. 260:3030). A major advantage of thisapproach is the fact that ribozymes are sequence-specific.

There are two basic types of ribozymes, namely, tetrahymena-type(Hasselhoff, 1988, Nature 334:585) and hammerhead-type. Tetrahymena-typeribozymes recognize sequences which are four bases in length, whilehammerhead-type ribozymes recognize base sequences 11-18 bases inlength. The longer the sequence, the greater the likelihood that thesequence will occur exclusively in the target mRNA species.Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating specific mRNA species, and18-base recognition sequences are preferable to shorter recognitionsequences which may occur randomly within various unrelated mRNAmolecules.

Ribozymes useful for inhibiting the expression of a regulator may bedesigned by incorporating target sequences into the basic ribozymestructure which are complementary to the mRNA sequence of the desiredregulator of the present invention, including but are not limited to,CCR10 or any component of the signal transduction pathway associatedwith CCR10 or a functional equivalent thereof. Ribozymes targeting thedesired regulator may be synthesized using commercially availablereagents (Applied Biosystems, Inc., Foster City, Calif.) or they may begenetically expressed from DNA encoding them.

In another aspect of the invention, the regulator can be inhibited byway of inactivating and/or sequestering the regulator. As such,inhibiting the effects of a regulator can be accomplished by using atransdominant negative mutant. Alternatively an antibody specific forthe desired regulator, otherwise known as an antagonist to the regulatormay be used. In one embodiment, the antagonist is a protein and/orcompound having the desirable property of interacting with a bindingpartner of the regulator and thereby competing with the correspondingwild-type regulator. In another embodiment, the antagonist is a proteinand/or compound having the desirable property of interacting with theregulator and thereby sequestering the regulator.

Antibodies

As will be understood by one skilled in the art, any antibody that canrecognize and bind to an antigen of interest is useful in the presentinvention. That is, the antibody can inhibit CCR10 or its ligand CCL27or any component of the signal transduction pathway associated withCCR10 or a functional equivalent thereof to provide a beneficial effect.

Methods of making and using antibodies are well known in the art. Forexample, polyclonal antibodies useful in the present invention aregenerated by immunizing rabbits according to standard immunologicaltechniques well-known in the art (see, e.g., Harlow et al., 1988, In:Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.). Suchtechniques include immunizing an animal with a chimeric proteincomprising a portion of another protein such as a maltose bindingprotein or glutathione (GSH) tag polypeptide portion, and/or a moietysuch that the antigenic protein of interest is rendered immunogenic(e.g., an antigen of interest conjugated with keyhole limpet hemocyanin,KLH) and a portion comprising the respective antigenic protein aminoacid residues. The chimeric proteins are produced by cloning theappropriate nucleic acids encoding the marker protein into a plasmidvector suitable for this purpose, such as but not limited to, pMAL-2 orpCMX.

However, the invention should not be construed as being limited solelyto methods and compositions including these antibodies or to theseportions of the antigens. Rather, the invention should be construed toinclude other antibodies, as that term is defined elsewhere herein, toantigens, or portions thereof. Further, the present invention should beconstrued to encompass antibodies, inter alia, bind to the specificantigens of interest, and they are able to bind the antigen present onWestern blots, in solution in enzyme linked immunoassays, influorescence activated cells sorting (FACS) assays, in magenetic-activedcell sorting (MACS) assays, and in immunofluorescence microscopy of acell transiently transfected with a nucleic acid encoding at least aportion of the antigenic protein, for example.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the antibody can specifically bind with anyportion of the antigen and the full-length protein can be used togenerate antibodies specific therefor. However, the present invention isnot limited to using the full-length protein as an immunogen. Rather,the present invention includes using an immunogenic portion of theprotein to produce an antibody that specifically binds with a specificantigen. That is, the invention includes immunizing an animal using animmunogenic portion, or antigenic determinant, of the antigen.

Once armed with the sequence of a specific antigen of interest and thedetailed analysis localizing the various conserved and non-conserveddomains of the protein, the skilled artisan would understand, based uponthe disclosure provided herein, how to obtain antibodies specific forthe various portions of the antigen using methods well-known in the artor to be developed.

The skilled artisan would appreciate, based upon the disclosure providedherein, that that present invention includes use of a single antibodyrecognizing a single antigenic epitope but that the invention is notlimited to use of a single antibody. Instead, the invention encompassesuse of at least one antibody where the antibodies can be directed to thesame or different antigenic protein epitopes.

The generation of polyclonal antibodies is accomplished by inoculatingthe desired animal with the antigen and isolating antibodies whichspecifically bind the antigen therefrom using standard antibodyproduction methods such as those described in, for example, Harlow etal. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor,N.Y.).

Monoclonal antibodies directed against full length or peptide fragmentsof a protein or peptide may be prepared using any well-known monoclonalantibody preparation procedures, such as those described, for example,in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold SpringHarbor, N.Y.) and in Tuszynski et al. (1988, Blood, 72:109-115).Quantities of the desired peptide may also be synthesized using chemicalsynthesis technology. Alternatively, DNA encoding the desired peptidemay be cloned and expressed from an appropriate promoter sequence incells suitable for the generation of large quantities of peptide.Monoclonal antibodies directed against the peptide are generated frommice immunized with the peptide using standard procedures as referencedherein.

Nucleic acid encoding the monoclonal antibody obtained using theprocedures described herein may be cloned and sequenced using technologywhich is available in the art, and is described, for example, in Wrightet al. (1992, Critical Rev. Immunol. 12:125-168), and the referencescited therein. Further, the antibody of the invention may be “humanized”using the technology described in, for example, Wright et al., and inthe references cited therein, and in Gu et al. (1997, Thrombosis andHematocyst 77:755-759), and other methods of humanizing antibodieswell-known in the art or to be developed.

The present invention also includes the use of humanized antibodiesspecifically reactive with epitopes of an antigen of interest. Thehumanized antibodies of the invention have a human framework and haveone or more complementarity determining regions (CDRs) from an antibody,typically a mouse antibody, specifically reactive with an antigen ofinterest. When the antibody used in the invention is humanized, theantibody may be generated as described in Queen, et al. (U.S. Pat. No.6,180,370), Wright et al., (supra) and in the references cited therein,or in Gu et al. (1997, Thrombosis and Hematocyst 77(4):755-759). Themethod disclosed in Queen et al. is directed in part toward designinghumanized immunoglobulins that are produced by expressing recombinantDNA segments encoding the heavy and light chain complementaritydetermining regions (CDRs) from a donor immunoglobulin capable ofbinding to a desired antigen, such as an epitope on an antigen ofinterest, attached to DNA segments encoding acceptor human frameworkregions. Generally speaking, the invention in the Queen patent hasapplicability toward the design of substantially any humanizedimmunoglobulin. Queen explains that the DNA segments will typicallyinclude an expression control DNA sequence operably linked to thehumanized immunoglobulin coding sequences, includingnaturally-associated or heterologous promoter regions. The expressioncontrol sequences can be eukaryotic promoter systems in vectors capableof transforming or transfecting eukaryotic host cells or the expressioncontrol sequences can be prokaryotic promoter systems in vectors capableof transforming or transfecting prokaryotic host cells. Once the vectorhas been incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the introducednucleotide sequences and as desired the collection and purification ofthe humanized light chains, heavy chains, light/heavy chain dimers orintact antibodies, binding fragments or other immunoglobulin forms mayfollow (Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NewYork, (1979), which is incorporated herein by reference).

The invention also includes functional equivalents of the antibodiesdescribed herein. Functional equivalents have binding characteristicscomparable to those of the antibodies, and include, for example,hybridized and single chain antibodies, as well as fragments thereof.Methods of producing such functional equivalents are disclosed in PCTApplication WO 93/21319 and PCT Application WO 89/09622.

Functional equivalents include polypeptides with amino acid sequencessubstantially the same as the amino acid sequence of the variable orhypervariable regions of the antibodies. “Substantially the same” aminoacid sequence is defined herein as a sequence with at least 70%,preferably at least about 80%, more preferably at least about 90%, evenmore preferably at least about 95%, and most preferably at least 99%homology to another amino acid sequence (or any integer in between 70and 99), as determined by the FASTA search method in accordance withPearson and Lipman, 1988 Proc. Nat'l. Acad. Sci. USA 85: 2444-2448.Chimeric or other hybrid antibodies have constant regions derivedsubstantially or exclusively from human antibody constant regions andvariable regions derived substantially or exclusively from the sequenceof the variable region of a monoclonal antibody from each stablehybridoma.

Single chain antibodies (scFv) or Fv fragments are polypeptides thatconsist of the variable region of the heavy chain of the antibody linkedto the variable region of the light chain, with or without aninterconnecting linker. Thus, the Fv comprises an antibody combiningsite.

Functional equivalents of the antibodies of the invention furtherinclude fragments of antibodies that have the same, or substantially thesame, binding characteristics to those of the whole antibody. Suchfragments may contain one or both Fab fragments or the F(ab′)₂ fragment.The antibody fragments contain all six complement determining regions ofthe whole antibody, although fragments containing fewer than all of suchregions, such as three, four or five complement determining regions, arealso functional. The functional equivalents are members of the IgGimmunoglobulin class and subclasses thereof, but may be or may combinewith any one of the following immunoglobulin classes: IgM, IgA, IgD, orIgE, and subclasses thereof. Heavy chains of various subclasses, such asthe IgG subclasses, are responsible for different effector functions andthus, by choosing the desired heavy chain constant region, hybridantibodies with desired effector function are produced. Exemplaryconstant regions are gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), andgamma 4 (IgG4). The light chain constant region can be of the kappa orlambda type.

The immunoglobulins of the present invention can be monovalent, divalentor polyvalent. Monovalent immunoglobulins are dimers (HL) formed of ahybrid heavy chain associated through disulfide bridges with a hybridlight chain. Divalent immunoglobulins are tetramers (H₂L₂) formed of twodimers associated through at least one disulfide bridge.

Antagonists

Inhibitors of the invention can be any agent that inhibits any componentof the CCR10/ligand axis. For example, the inhibitor of the inventionincludes, but is not limited to, a molecule that has an affinity forbinding to CCR10 and/or CCL27 and inhibiting the CCR10:CCL27 pathway(e.g., CCR10/ligand axis).

In one embodiment, the invention provides inhibitors of the CCR10/ligandaxis by blocking the interaction between CCR10 and its ligand CCL27. Theblockage of the interaction between CCR10 and CCL27 can be accomplishedusing a ligand antagonist or receptor antagonist. Such may be ligandmutein antagonists, antibody antagonists to ligand or receptor, ordrugs, e.g., small molecules, which block the chemoattraction betweenCCR10 and CCL27.

In one embodiment, the invention provides antagonists that block thesignaling and/or effector biology of the CCR10/ligand axis in order toenhance the immune response. Antagonist activity may be tested orscreened for using well known methods. Tests for ability to antagonizechemokine binding or chemoattractant activity can be developed. Variousligand homologs can be created which retain receptor binding capacity,but lack signaling capability, thus serving as competitive bindingmolecules. Small molecules may also be screened for ability toantagonize chemokine function, e.g., chemoattraction, receptor binding,and other effects mediated by chemokine.

Other binding compositions, which will often have similar uses, includemolecules that bind with specificity to the receptor (e.g., CCR10) orthe chemokine (e.g., CCL27), in a binding partner-binding partnerfashion, an antibody-antigen interaction, a ligand:receptor interactionwith or without signaling, or in a natural physiologically relevantprotein-protein interaction, either covalent or non-covalent, e.g.,proteins which specifically associate with chemokine receptor protein.The molecule may be a polymer, or chemical reagent. A functional analogmay be a protein with structural modifications, or may be a structurallyunrelated molecule, e.g., which has a molecular shape which interactswith the appropriate binding determinants.

Drug screening can be performed to identify compounds having capacity tobind to receptor, and/or to block chemoattraction to chemokine or thenatural interaction with ligand. Subsequent biological assays can thenbe utilized to determine if the compound has intrinsic binding orblocking activity, e.g., an antagonist. Mutein antagonists may bedeveloped which maintain receptor binding but lack signaling.

Structural studies of the ligands will lead to design of new variants,particularly analogs exhibiting antagonist properties on the receptor.This can be combined with previously described screening methods toisolate muteins exhibiting desired spectra of activities. Or ligands maybe used to target or label receptor bearing cells.

As receptor specific binding molecules are provided, also included aresmall molecules identified by screening procedures. In particular, it iswell known in the art how to screen for small molecules which interfere,e.g., with ligand binding to the receptor, often by specific binding tothe receptor and blocking of binding by natural ligand. Such moleculesmay compete with natural ligands, and selectively bind to the respectivechemokines (e.g., CCL27) or CCR10 receptor. Similarly, assays may bedeveloped which can screen for blockage of downstream signaling pathwaysof the chemokine signaling pathways.

Activators

In various embodiments, the present invention includes compositions andmethods of treating an autoimmune and inflammatory disorders including,but are not limited to, arthritic diseases such as rheumatoid arthritis,osteoarthritis, gouty arthritis, spondylitis; Behcet disease; sepsis,septic shock, endotoxic shock, gram negative sepsis, gram positivesepsis, and toxic shock syndrome; multiple organ injury syndromesecondary to septicemia, trauma, or hemorrhage; ophthalmic disorderssuch as allergic conjunctivitis, vernal conjunctivitis, uveitis, andthyroid-associated ophthalmopathy; eosinophilic granuloma; pulmonary orrespiratory disorders such as asthma, chronic bronchitis, allergicrhinitis, ARDS, chronic pulmonary inflammatory disease (e.g., chronicobstructive pulmonary disease), silicosis, pulmonary sarcoidosis,pleurisy, alveolitis, vasculitis, pneumonia, bronchiectasis, andpulmonary oxygen toxicity; reperfusion injury of the myocardium, brain,or extremities; fibrosis such as cystic fibrosis; keloid formation orscar tissue formation; atherosclerosis; autoimmune diseases such assystemic lupus erythematosus (SLE), autoimmune thyroiditis, multiplesclerosis, some forms of diabetes, and Reynaud's syndrome; connectivetissue disease, autoimmune pulmonary inflammation, Guillain Banesyndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis,myasthenia gravis, graft versus host disease and autoimmune inflammatoryeye disease; transplant rejection disorders such as GVHD and allograftrejection, chronic glomerulonephritis; inflammatory bowel diseases suchas Crohn's disease, ulcerative colitis and necrotizing enterocolitis,inflammatory dermatoses such as contact dermatitis, atopic dermatitis,psoriasis, or urticarial; autoimmune intestinal inflammation; fever andmyalgias due to infection; central or peripheral nervous systeminflammatory disorders such as meningitis, encephalitis, and brain orspinal cord injury due to minor trauma; Sjorgren's syndrome; diseasesinvolving leukocyte diapedesis; alcoholic hepatitis; bacterialpneumonia; antigen-antibody complex mediated diseases; hypovolemicshock; Type diabetes mellitus; acute and delayed hypersensitivity;disease states due to leukocyte dyscrasia and metastasis; thermalinjury; granulocyte transfusion associated syndromes; cytokine-inducedtoxicity; and allergic reactions and conditions (e.g., anaphylaxis,serum sickness, drug reactions, food allergies, insect venom allergies,mastocytosis, allergic rhinitis, hypersensitivity pneumonitis,urticaria, angioedema, eczema, atopic dermatitis, allergic contactdermatitis, erythema multiform, Stevens Johnson syndrome, allergicconjunctivitis, atopic keratoconjunctivitis, venerealkeratoconjunctivitis, giant papillary conjunctivitis and contactallergies), such as asthma (particularly allergic asthma) or otherrespiratory problems.

It will be understood by one skilled in the art, based upon thedisclosure provided herein, that an increase in the level of any one ormore of CCR10, CCR10/ligand axis, and their functional equivalentsencompasses the increase in expression, including transcription,translation, or both of any one or more of CCR10, CCR10/ligand axis, andtheir functional equivalents. The skilled artisan will also appreciate,once armed with the teachings of the present invention, that an increasein the level of any one or more of CCR10, CCR10/ligand axis, and theirfunctional equivalents includes an increase in activity of any one ormore of CCR10, CCR10/ligand axis, and their functional equivalents.Thus, increasing the level or activity of any one or more of CCR10,CCR10/ligand axis, and their functional equivalents includes, but is notlimited to, increasing the amount of polypeptide, increasingtranscription, translation, or both, of a nucleic acid encoding one ormore of CCR10, CCR10/ligand axis, and their functional equivalents; andit also includes increasing any activity of any one or more of CCR10,CCR10/ligand axis, and their functional equivalents as well.

Thus, the present invention relates to the prevention and treatment of adesired autoimmune or inflammatory disease or disorder by administrationof a polypeptide, a recombinant polypeptide, an active polypeptidefragment, or an activator of expression or activity of one or more ofCCR10, CCR10/ligand axis, and their functional equivalents.

It is understood by one skilled in the art, that an increase in thelevel of, for example, CCR10 encompasses the increase of CCR10 proteinexpression. Additionally, the skilled artisan would appreciate, that anincrease in the level of CCR10 includes an increase in CCR10 activity.Thus, increasing the level or activity of CCR10 includes, but is notlimited to, increasing transcription, translation, or both, of a nucleicacid encoding CCR10; and it also includes increasing any activity ofCCR10 as well.

Activation of CCR10 can be assessed using a wide variety of methods,including those disclosed herein, as well as methods well-known in theart or to be developed in the future. That is, the routineer wouldappreciate, based upon the disclosure provided herein, that increasingthe level or activity of CCR10 can be readily assessed using methodsthat assess the level of a nucleic acid encoding CCR10 (e.g., mRNA)and/or the level of CCR10 polypeptide in a biological sample obtainedfrom a subject.

A CCR10 activator can include, but should not be construed as beinglimited to, a chemical compound, a protein, a peptidomemetic, anantibody, a nucleic acid molecule. One of skill in the art would readilyappreciate, based on the disclosure provided herein, that a CCR10activator encompasses a chemical compound that increases the level,enzymatic activity, or the like of CCR10. Additionally, a CCR10activator encompasses a chemically modified compound, and derivatives,as is well known to one of skill in the chemical arts.

It will be understood by one skilled in the art, based upon thedisclosure provided herein, that an increase in the level of CCR10encompasses the increase in CCR10 expression, including transcription,translation, or both. The skilled artisan will also appreciate, oncearmed with the teachings of the present invention, that an increase inthe level of CCR10 includes an increase in CCR10 activity (e.g.,enzymatic activity, receptor binding activity, etc.). Thus, increasingthe level or activity of CCR10 includes, but is not limited to,increasing the amount of CCR10 polypeptide, increasing transcription,translation, or both, of a nucleic acid encoding CCR10; and it alsoincludes increasing any activity of a CCR10 polypeptide as well. TheCCR10 activator compositions and methods of the invention canselectively activate CCR10.

Further, one of skill in the art would, when equipped with thisdisclosure and the methods exemplified herein, appreciate that a CCR10activator includes such activators as discovered in the future, as canbe identified by well-known criteria in the art of pharmacology, such asthe physiological results of activation of CCR10 as described in detailherein and/or as known in the art. Therefore, the present invention isnot limited in any way to any particular CCR10 activator as exemplifiedor disclosed herein; rather, the invention encompasses those activatorsthat would be understood by the routineer to be useful as are known inthe art and as are discovered in the future.

Further methods of identifying and producing a CCR10 activator are wellknown to those of ordinary skill in the art, including, but not limited,obtaining an activator from a naturally occurring source. Alternatively,a CCR10 activator can be synthesized chemically. Further, the routineerwould appreciate, based upon the teachings provided herein, that a CCR10activator can be obtained from a recombinant organism. Compositions andmethods for chemically synthesizing CCR10 activators and for obtainingthem from natural sources are well known in the art and are described inthe art.

One of skill in the art will appreciate that an activator can beadministered as a small molecule chemical, a protein, a nucleic acidconstruct encoding a protein, or combinations thereof. Numerous vectorsand other compositions and methods are well known for administering aprotein or a nucleic acid construct encoding a protein to cells ortissues. Therefore, the invention includes a method of administering aprotein or a nucleic acid encoding a protein that is an activator ofCCR10.

One of skill in the art will realize that diminishing the amount oractivity of a molecule that itself diminishes the amount or activity ofCCR10 can serve to increase the amount or activity of CCR10. Anyinhibitor of a regulator of CCR10 is encompassed in the invention. As anon-limiting example, antisense is described as a form of inhibiting aregulator of CCR10 in order to increase the amount or activity of CCR10.Antisense oligonucleotides are DNA or RNA molecules that arecomplementary to some portion of a mRNA molecule. When present in acell, antisense oligonucleotides hybridize to an existing mRNA moleculeand inhibit translation into a gene product Inhibiting the expression ofa gene using an antisense oligonucleotide is well known in the art(Marcus-Sekura, 1988, Anal. Biochem. 172:289), as are methods ofexpressing an antisense oligonucleotide in a cell (Inoue, U.S. Pat. No.5,190,931). The methods of the invention include the use of antisenseoligonucleotide to diminish the amount of a molecule that causes adecrease in the amount or activity CCR10, thereby increasing the amountor activity of CCR10. Contemplated in the present invention areantisense oligonucleotides that are synthesized and provided to the cellby way of methods well known to those of ordinary skill in the art. Asan example, an antisense oligonucleotide can be synthesized to bebetween about 10 and about 100, more preferably between about 15 andabout 50 nucleotides long. The synthesis of nucleic acid molecules iswell known in the art, as is the synthesis of modified antisenseoligonucleotides to improve biological activity in comparison tounmodified antisense oligonucleotides (Tullis, 1991, U.S. Pat. No.5,023,243).

Similarly, the expression of a gene may be inhibited by thehybridization of an antisense molecule to a promoter or other regulatoryelement of a gene, thereby affecting the transcription of the gene.Methods for the identification of a promoter or other regulatory elementthat interacts with a gene of interest are well known in the art, andinclude such methods as the yeast two hybrid system (Bartel and Fields,eds., In: The Yeast Two Hybrid System, Oxford University Press, Cary,N.C.).

Alternatively, inhibition of a gene expressing a protein that diminishesthe level or activity of CCR10 can be accomplished through the use of aribozyme. Using ribozymes for inhibiting gene expression is well knownto those of skill in the art (see, e.g., Cech et al., 1992, J. Biol.Chem. 267:17479; Hampel et al., 1989, Biochemistry 28: 4929; Altman etal., U.S. Pat. No. 5,168,053). Ribozymes are catalytic RNA moleculeswith the ability to cleave other single-stranded RNA molecules.Ribozymes are known to be sequence specific, and can therefore bemodified to recognize a specific nucleotide sequence (Cech, 1988, J.Amer. Med. Assn. 260:3030), allowing the selective cleavage of specificmRNA molecules. Given the nucleotide sequence of the molecule, one ofordinary skill in the art could synthesize an antisense oligonucleotideor ribozyme without undue experimentation, provided with the disclosureand references incorporated herein.

One of skill in the art will appreciate that a CCR10 polypeptide, arecombinant CCR10 polypeptide, or an active CCR10 polypeptide fragmentcan be administered singly or in any combination thereof. Further, aCCR10 polypeptide, a recombinant CCR10 polypeptide, or an active CCR10polypeptide fragment can be administered singly or in any combinationthereof in a temporal sense, in that they may be administeredsimultaneously, before, and/or after each other. One of ordinary skillin the art will appreciate, based on the disclosure provided herein,that a CCR10 polypeptide, a recombinant CCR10 polypeptide, or an activeCCR10 polypeptide fragment can be used to prevent or treat aneurodegenerative disease or disorder, and that an activator can be usedalone or in any combination with another CCR10 polypeptide, recombinantCCR10 polypeptide, active CCR10 polypeptide fragment, or CCR10 activatorto effect a therapeutic result.

One of skill in the art, when armed with the disclosure herein, wouldappreciate that the treating a neurodegenerative disease or disorderencompasses administering to a subject a CCR10 polypeptide, arecombinant CCR10 polypeptide, an active CCR10 polypeptide fragment, orCCR10 activator as a preventative measure against a neurodegenerativedisease or disorder. As more fully discussed elsewhere herein, methodsof increasing the level or activity of a CCR10 encompass a wide plethoraof techniques for increasing not only CCR10 activity, but also forincreasing expression of a nucleic acid encoding CCR10. Additionally, asdisclosed elsewhere herein, one skilled in the art would understand,once armed with the teaching provided herein, that the present inventionencompasses a method of preventing a wide variety of diseases whereincreased expression and/or activity of CCR10 mediates, treats orprevents the disease. Further, the invention encompasses treatment orprevention of such diseases discovered in the future.

The invention encompasses administration of a CCR10 polypeptide, arecombinant CCR10 polypeptide, an active CCR10 polypeptide fragment, ora CCR10 activator to practice the methods of the invention; the skilledartisan would understand, based on the disclosure provided herein, howto formulate and administer the appropriate CCR10 polypeptide,recombinant CCR10 polypeptide, active CCR10 polypeptide fragment, orCCR10 activator to a subject. However, the present invention is notlimited to any particular method of administration or treatment regimen.This is especially true where it would be appreciated by one skilled inthe art, equipped with the disclosure provided herein, including thereduction to practice using an art-recognized model of an autoimmunedisease, that methods of administering a CCR10 polypeptide, arecombinant CCR10 polypeptide, an active CCR10 polypeptide fragment, orCCR10 activator can be determined by one of skill in the pharmacologicalarts.

As used herein, the term “pharmaceutically-acceptable carrier” means achemical composition with which an appropriate CCR10 polypeptide,recombinant CCR10 polypeptide, active CCR10 polypeptide fragment, orCCR10 activator, may be combined and which, following the combination,can be used to administer the appropriate CCR10 polypeptide, recombinantCCR10 polypeptide, active CCR10 polypeptide fragment, or CCR10 activatorto a subject.

Modification of Nucleic Acid Molecules

Inhibition of CCR10 or any component of the signal transduction pathwayassociated with CCR10 or a functional equivalent thereof can beaccomplished using a nucleic acid molecule. For example, the inhibitoris selected from the group consisting of a small interfering RNA(siRNA), a microRNA, an antisense nucleic acid, a ribozyme, anexpression vector encoding a transdominant negative mutant, and thelikes.

By way of example, modification of nucleic acid molecules is describedin the context of an siRNA molecule. However, the methods of modifyingnucleic acid molecules can be applied to other types of nucleic acidbased modulators of the invention.

Polynucleotides of the siRNA may be prepared using any of a variety oftechniques, which are useful for the preparation of specifically desiredsiRNA polynucleotides. For example, a polynucleotide may be amplifiedfrom a cDNA prepared from a suitable cell or tissue type. Such apolynucleotide may be amplified via polymerase chain reaction (PCR).Using this approach, sequence-specific primers are designed based on thesequences provided herein, and may be purchased or synthesized directly.An amplified portion of the primer may be used to isolate a full-lengthgene, or a desired portion thereof, from a suitable DNA library usingwell known techniques. A library (cDNA or genomic) is screened using oneor more polynucleotide probes or primers suitable for amplification.Preferably, the library is size-selected to include largerpolynucleotide sequences. Random primed libraries may also be preferredin order to identify 5′ and other upstream regions of the genes. Genomiclibraries are preferred for obtaining introns and extending 5′sequences. The siRNA polynucleotide contemplated by the presentinvention may also be selected from a library of siRNA polynucleotidesequences.

For hybridization techniques, a partial polynucleotide sequence may belabeled (e.g., by nick-translation or end-labeling with ³²P) using wellknown techniques. A bacterial or bacteriophage library may then bescreened by hybridization to filters containing denatured bacterialcolonies (or lawns containing phage plaques) with the labeled probe(see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 2001).Hybridizing colonies or plaques are selected and expanded, and the DNAis isolated for further analysis.

Alternatively, numerous amplification techniques are known in the artfor obtaining a full-length coding sequence from a partial cDNAsequence. Within such techniques, amplification is generally performedvia PCR. One such technique is known as “rapid amplification of cDNAends” or RACE (see, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor,N.Y., 2001).

A number of specific siRNA polynucleotide sequences useful forinterfering with target polypeptide expression are presented in theExamples, the Drawings, and in the Sequence Listing included herein.siRNA polynucleotides may generally be prepared by any method known inthe art, including, for example, solid phase chemical synthesis.Modifications in a polynucleotide sequence may also be introduced usingstandard mutagenesis techniques, such as oligonucleotide-directedsite-specific mutagenesis. Further, siRNAs may be chemically modified orconjugated with other molecules to improve their stability and/ordelivery properties. Included as one aspect of the invention are siRNAsas described herein, wherein one or more ribose sugars has been removedtherefrom.

Alternatively, siRNA polynucleotide molecules may be generated by invitro or in vivo transcription of suitable DNA sequences (e.g.,polynucleotide sequences encoding a target polypeptide, or a desiredportion thereof), provided that the DNA is incorporated into a vectorwith a suitable RNA polymerase promoter (such as for example, T7, U6,H1, or SP6 although other promoters may be equally useful). In addition,an siRNA polynucleotide may be administered to a mammal, as may be a DNAsequence (e.g., a recombinant nucleic acid construct as provided herein)that supports transcription (and optionally appropriate processingsteps) such that a desired siRNA is generated in vivo.

In one embodiment, an siRNA polynucleotide, wherein the siRNApolynucleotide is capable of interfering with expression of a targetpolypeptide can be used to generate a silenced cell. Any siRNApolynucleotide that, when contacted with a biological source for aperiod of time, results in a significant decrease in the expression ofthe target polypeptide is included in the invention. Preferably thedecrease is greater than about 10%, more preferably greater than about20%, more preferably greater than about 30%, more preferably greaterthan about 40%, about 50%, about 60%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95% or about 98% relative to the expressionlevel of the target polypeptide detected in the absence of the siRNA.Preferably, the presence of the siRNA polynucleotide in a cell does notresult in or cause any undesired toxic effects, for example, apoptosisor death of a cell in which apoptosis is not a desired effect of RNAinterference.

In another embodiment, the siRNA polynucleotide that, when contactedwith a biological source for a period of time, results in a significantdecrease in the expression of the target polypeptide. Preferably thedecrease is about 10%-20%, more preferably about 20%-30%, morepreferably about 30%-40%, more preferably about 40%-50%, more preferablyabout 50%-60%, more preferably about 60%-70%, more preferably about70%-80%, more preferably about 80%-90%, more preferably about 90%-95%,more preferably about 95%-98% relative to the expression level of thetarget polypeptide detected in the absence of the siRNA. Preferably, thepresence of the siRNA polynucleotide in a cell does not result in orcause any undesired toxic effects.

In yet another embodiment, the siRNA polynucleotide that, when contactedwith a biological source for a period of time, results in a significantdecrease in the expression of the target polypeptide. Preferably thedecrease is about 10% or more, more preferably about 20% or more, morepreferably about 30% or more, more preferably about 40% or more, morepreferably about 50% or more, more preferably about 60% or more, morepreferably about 70% or more, more preferably about 80% or more, morepreferably about 90% or more, more preferably about 95% or more, morepreferably about 98% or more relative to the expression level of thetarget polypeptide detected in the absence of the siRNA. Preferably, thepresence of the siRNA polynucleotide in a cell does not result in orcause any undesired toxic effects.

Any polynucleotide of the invention may be further modified to increaseits stability in vivo. Possible modifications include, but are notlimited to, the addition of flanking sequences at the 5′ and/or 3′ ends;the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterlinkages in the backbone; and/or the inclusion of nontraditional basessuch as inosine, queosine, and wybutosine and the like, as well asacetyl-methyl-, thio- and other modified forms of adenine, cytidine,guanine, thymine, and uridine.

Genetic Modification

In other related aspects, the invention includes an isolated nucleicacid encoding a modulator of the invention, wherein the nucleic acid isoperably linked to a promoter/regulatory sequence such that the nucleicacid is preferably capable of directing expression of the proteinencoded by the nucleic acid. Thus, the invention encompasses expressionvectors and methods for the introduction of exogenous DNA into cellswith concomitant expression of the exogenous DNA in the cells such asthose described, for example, in Sambrook et al. (2001, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York),and in Ausubel et al. (1997, Current Protocols in Molecular Biology,John Wiley & Sons, New York).

The desired polynucleotide can be cloned into a number of types ofvectors. However, the present invention should not be construed to belimited to any particular vector. Instead, the present invention shouldbe construed to encompass a wide plethora of vectors which are readilyavailable and/or well-known in the art. For example, a desiredpolynucleotide of the invention can be cloned into a vector including,but not limited to a plasmid, a phagemid, a phage derivative, an animalvirus, and a cosmid. Vectors of particular interest include expressionvectors, replication vectors, probe generation vectors, and sequencingvectors.

In specific embodiments, the expression vector is selected from thegroup consisting of a viral vector, a bacterial vector and a mammaliancell vector. Numerous expression vector systems exist that comprise atleast a part or all of the compositions discussed above. Prokaryote-and/or eukaryote-vector based systems can be employed for use with thepresent invention to produce polynucleotides, or their cognatepolypeptides. Many such systems are commercially and widely available.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001), and in Ausubel et al.(1997), and in other virology and molecular biology manuals. Viruses,which are useful as vectors include, but are not limited to,retroviruses, adenoviruses, adeno-associated viruses, herpes viruses,and lentiviruses. In general, a suitable vector contains an origin ofreplication functional in at least one organism, a promoter sequence,convenient restriction endonuclease sites, and one or more selectablemarkers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No.6,326,193.

For expression of the desired polynucleotide, at least one module ineach promoter functions to position the start site for RNA synthesis.The best known example of this is the TATA box, but in some promoterslacking a TATA box, such as the promoter for the mammalian terminaldeoxynucleotidyl transferase gene and the promoter for the SV40 genes, adiscrete element overlying the start site itself helps to fix the placeof initiation.

Additional promoter elements, i.e., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either co-operativelyor independently to activate transcription.

A promoter may be one naturally associated with a gene or polynucleotidesequence, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment and/or exon. Such a promoter canbe referred to as “endogenous.” Similarly, an enhancer may be onenaturally associated with a polynucleotide sequence, located eitherdownstream or upstream of that sequence. Alternatively, certainadvantages will be gained by positioning the coding polynucleotidesegment under the control of a recombinant or heterologous promoter,which refers to a promoter that is not normally associated with apolynucleotide sequence in its natural environment. A recombinant orheterologous enhancer refers also to an enhancer not normally associatedwith a polynucleotide sequence in its natural environment. Suchpromoters or enhancers may include promoters or enhancers of othergenes, and promoters or enhancers isolated from any other prokaryotic,viral, or eukaryotic cell, and promoters or enhancers not “naturallyoccurring,” i.e., containing different elements of differenttranscriptional regulatory regions, and/or mutations that alterexpression. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (U.S. Pat. No.4,683,202, U.S. Pat. No. 5,928,906). Furthermore, it is contemplated thecontrol sequences that direct transcription and/or expression ofsequences within non-nuclear organelles such as mitochondria,chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in the celltype, organelle, and organism chosen for expression. Those of skill inthe art of molecular biology generally know how to use promoters,enhancers, and cell type combinations for protein expression, forexample, see Sambrook et al. (2001). The promoters employed may beconstitutive, tissue-specific, inducible, and/or useful under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins and/or peptides. The promoter may be heterologousor endogenous.

A promoter sequence exemplified in the experimental examples presentedherein is the immediate early cytomegalovirus (CMV) promoter sequence.This promoter sequence is a strong constitutive promoter sequencecapable of driving high levels of expression of any polynucleotidesequence operatively linked thereto. However, other constitutivepromoter sequences may also be used, including, but not limited to thesimian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV),human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter,Moloney virus promoter, the avian leukemia virus promoter, Epstein-Barrvirus immediate early promoter, Rous sarcoma virus promoter, as well ashuman gene promoters such as, but not limited to, the actin promoter,the myosin promoter, the hemoglobin promoter, and the muscle creatinepromoter. Further, the invention should not be limited to the use ofconstitutive promoters. Inducible promoters are also contemplated aspart of the invention. The use of an inducible promoter in the inventionprovides a molecular switch capable of turning on expression of thepolynucleotide sequence which it is operatively linked when suchexpression is desired, or turning off the expression when expression isnot desired. Examples of inducible promoters include, but are notlimited to a metallothionine promoter, a glucocorticoid promoter, aprogesterone promoter, and a tetracycline promoter. Further, theinvention includes the use of a tissue specific promoter, which promoteris active only in a desired tissue. Tissue specific promoters are wellknown in the art and include, but are not limited to, the HER-2 promoterand the PSA associated promoter sequences.

In order to assess the expression of the modulator, the expressionvector to be introduced into a cell can also contain either a selectablemarker gene or a reporter gene or both to facilitate identification andselection of expressing cells from the population of cells sought to betransfected or infected through viral vectors. In other embodiments, theselectable marker may be carried on a separate piece of DNA and used ina co-transfection procedure. Both selectable markers and reporter genesmay be flanked with appropriate regulatory sequences to enableexpression in the host cells. Useful selectable markers are known in theart and include, for example, antibiotic-resistance genes, such as neoand the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Reportergenes that encode for easily assayable proteins are well known in theart. In general, a reporter gene is a gene that is not present in orexpressed by the recipient organism or tissue and that encodes a proteinwhose expression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells.

Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (see, e.g.,Ui-Tei et al., 2000 FEBS Lett. 479:79-82). Suitable expression systemsare well known and may be prepared using well known techniques orobtained commercially. Internal deletion constructs may be generatedusing unique internal restriction sites or by partial digestion ofnon-unique restriction sites. Constructs may then be transfected intocells that display high levels of modulator polynucleotide and/orpolypeptide expression. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast or insectcell by any method in the art. For example, the expression vector can betransferred into a host cell by physical, chemical or biological means.It is readily understood that the introduction of the expression vectorcomprising the polynucleotide of the invention yields a silenced cellwith respect to a regulator.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York), and in Ausubel et al. (1997, Current Protocols in MolecularBiology, John Wiley & Sons, New York).

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Apreferred colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (i.e., an artificial membrane vesicle). Thepreparation and use of such systems is well known in the art.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

Any DNA vector or delivery vehicle can be utilized to transfer thedesired polynucleotide to a cell in vitro or in vivo. In the case wherea non-viral delivery system is utilized, a preferred delivery vehicle isa liposome. The above-mentioned delivery systems and protocols thereforecan be found in Gene Targeting Protocols, 2ed., pp 1-35 (2002) and GeneTransfer and Expression Protocols, Vol. 7, Murray ed., pp 81-89 (1991).

“Liposome” is a generic term encompassing a variety of single andmultilamellar lipid vehicles formed by the generation of enclosed lipidbilayers or aggregates. Liposomes may be characterized as havingvesicular structures with a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes have multiple lipid layersseparated by aqueous medium. They form spontaneously when phospholipidsare suspended in an excess of aqueous solution. The lipid componentsundergo self-rearrangement before the formation of closed structures andentrap water and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). However, the present invention also encompassescompositions that have different structures in solution than the normalvesicular structure. For example, the lipids may assume a micellarstructure or merely exist as nonuniform aggregates of lipid molecules.Also contemplated are lipofectamine-nucleic acid complexes.

Cell Populations

T helper cells (also known as effector T cells or Th cells) are asub-group of lymphocytes (a type of white blood cell or leukocyte) thatplays an important role in establishing and maximizing the capabilitiesof the immune system and in particular in activating and directing otherimmune cells. Different types of Th cells have been identified thatoriginate in outcome of a differentiation process and are associatedwith a specific phenotype. Following T cell development, matured, naive(meaning they have never been exposed to the antigen to which they canrespond) T cells leave the thymus and begin to spread throughout thebody. Naive T cells can differentiate into a T-helper 1 (Th1), T-helper2 (Th2), T-helper 17 (Th17) or regulatory T cell (Treg) phenotype.

Each of these Th cell types secretes cytokines, proteins or peptidesthat stimulate or interact with other leukocytes, including Th cells.However, each cell type has a peculiar phenotype and activity thatinterferes and often conflict with the other.

Th1, Th2, and Th17 (inflammatory T-helper or inflammatory Th), promoteinflammation responses trough secretion of pro-inflammatory cytokines,such as IL-1, IL-6, TNF-α, IL-17, IL21, IL23, and/or through activationand/or inhibition of other T cell including other Th cells (for exampleTh1 cell suppresses Th2 and Th17, Th2 suppresses Th1 and Th17). Tregsinstead, are a component of the immune system that suppresses biologicalactivities of other cells associated to an immune response. Inparticular, Tregs can secrete immunosuppressive cytokines TGF-β andInterleukin 10, and are known to be able to limit or suppressinflammation.

The present invention is based on the discovery of the important role ofCCR10 in a positive feedback circuit of immune homeostatic regulation inthe skin where CCR10 maintains balanced presence and function ofresident regulatory T (Treg) vs. effector T (Teff) cells, which in turnhelp to establish a homeostatic environment important for maintenance ofthe CCR10+ resident T cells.

Therapeutic Application

The present invention includes a modulator of CCR10 or any component ofthe signal transduction pathway associated with CCR10 or a functionalequivalent thereof. The present invention includes a method of enhancingthe immune response in a mammal comprising administering the mammal inneed thereof with an inhibitor of CCR10 or any component of the signaltransduction pathway associated with CCR10 or a functional equivalentthereof. In one embodiment, the present invention includes a method ofinhibiting the immune response in a mammal comprising administering themammal in need thereof with an activator of CCR10 or any component ofthe signal transduction pathway associated with CCR10 or a functionalequivalent thereof.

In one embodiment, the invention provides compositions and methods fortreating an autoimmune disease. In one embodiment, the autoimmunedisease is psoriasis. In another embodiment, the autoimmune disease isautoimmune intestinal inflammation. Therefore the present inventionincludes a therapeutic benefit of inhibiting the immune response in asubject by activating one or more of CCR10, CCR10/ligand axis, and theirfunctional equivalents.

In one embodiment, the mammal has a type of skin disease. In oneembodiment, the skin disease is associated with an inappropriate levelof immune response that is unable to alleviate the skin disease.Therefore, the invention can be used to increase the immune response inmammals that are in need of an enhancement of their immune response.Thus, treatment of the skin disease is accomplished by inducing anenhanced immune response using an inhibitor of CCR10 or any component ofthe signal transduction pathway associated with CCR10 or a functionalequivalent thereof of the invention.

Accordingly, the invention provides compositions and methods fortargeting one or more of CCR10, CCR10/ligand axis, and their functionalequivalents for enhancing an immune response in a subject. That is, theinvention is based on the novel discovery that CCR10 plays a role inregulating balanced maintenance and function of the skin-resident Tregand Teff cells to prevent overactive immune responses. Therefore, byinhibiting one or more of CCR10, CCR10/ligand axis, and their functionalequivalents, an immune response can be enhanced in a subject to treatdiseases and disorders that would be suppressed by the enhanced immuneresponse, including but not limited to skin-specific infections byparasites (e.g., protozoan and metazoan pathogens such as Leishmaniaspecies, Schistosoma species, Trypanosoma species), bacteria (e.g.,Staphylococcus aureus, MRSA-methicillin resistant staph aureus,Streptococcus pyogenes, Pseudomonas Aeruginosas, Corynebacteria,Mycobacteria), virus (e.g. Herpes simplex virus type 1, herpes simplexvirus type 2, Human Herpes Viruses, varicella-zoster virus, humanpapillomavirus, Coxsackie virus A16, Hepatitis B virus, Epstein Barrvirus), fungi (e.g., Candida species, Trichophyton, epidermophyton,microsporum, Sporothrix schenckii, Blastomycosis dermatitidis), andother microorganisms and associated symptoms such as Cutaneousleishmaniasis (Aleppo boil, Baghdad boil, Bay sore, Biskra button,Chiclero ulcer, Delhi boil, Kandahar sore, Lahore sore, Leishmaniasistropica, Oriental sore, Pian bois, Uta), Dracunculiasis (Dracontiasis,Guinea worm disease, Medina worm), Elephantiasis tropica (Elephantiasisarabum), Elephant skin, Human trypanosomiasis, Gnathostomiasis, Loa loafilariasis, Mucocutaneous leishmaniasis (Espundia, Leishmaniasisamericana), Post-kala-azar dermal leishmaniasis (Post-kala-azardermatosis), Swimmer's itch (Cercarial dermatitis, Schistosome cercarialdermatitis), Impetigo, Ecthyma, Abscess, Furuncles, and Carbuncles,Staphyloccocal Scalded Skin Syndrome, Toxic Shock Syndrome, Cellulitis,Ecthyma, Erysipelas, Scarlet Fever, Necrotizing Fasciitis, StreptococcalPeri-anal Disease, Streptococcal Toxic Shock Syndrome, Leprosy, SkinTuberculosis, Folliculitis, Cellulitis, Pyoderma, Rickettsial Disease,Spotted Fever, Lupus vulgaris, Scrofuloderma, Warty tuberculosis,Tuberculoid leprosy, Buruli ulcer, cold sores, genital herpes, Herpeszoster, Molluscum contagiosum, Warts, Hand, foot and mouth disease,papulovesicular acrodermatitis, Kaposi's sarcoma, Candidiasis,Dermatophytoses, onychomycosis. Sporotrichosis, Blastomycosis; andcancers such as Basal cell carcinoma, Squamous cell carcinoma, Kaposi'ssarcoma, Merkel cell carcinoma Sebaceous gland carcinoma, Mycosisfungoides, Cutaneous lymphoma, Skin adnexal tumor, African Kaposi'ssarcoma, Epidemic Kaposi's sarcoma.

As a non-limiting example, the mammal can have a skin disease associatedwith infection of a pathogenic organism. A pathogenic organism includesbut is not limited to a viruse, (e.g., single stranded RNA viruses,single stranded DNA viruses, double-stranded DNA viruses, HIV, hepatitisA, B, and C virus, HSV, CMV, EBV, HPV), a parasite (e.g., protozoan andmetazoan pathogens such as Plasmodia species, Leishmania species,Schistosoma species, Trypanosoma species), bacteria (e.g., Mycobacteria,Salmonella, Streptococci, E. coli, Staphylococci), fungi (e.g., Candidaspecies, Aspergillus species), Pneumocystis carinii, and the like.

Other types of infections include, but are not limited to, viralinfections, bacterial infections, anthrax, parasitic infections, fungalinfections and prion infection.

Viral infections include, but are not limited to, infections byhepatitis C, hepatitis B, influenza virus, herpes simplex virus (HSV),human immunodeficiency virus (HIV), respiratory syncytial virus (RSV),vesicular stomatitis virus (VSV), cytomegalovirus (CMV), poliovirus,encephalomyocarditis virus (EMCV), human papillomavirus (HPV) andsmallpox virus. In one embodiment, the infection is an upper respiratorytract infection caused by viruses and/or bacteria, in particular, flu,more specifically, bird flu.

Bacterial infections include, but are not limited to, infections bystreptococci, staphylococci, E. Coli, and pseudomonas. In oneembodiment, the bacterial infection is an intracellular bacterialinfection which is an infection by an intracellular bacterium such asmycobacteria (tuberculosis), chlamydia, mycoplasma, listeria, and afacultative intracelluar bacterium such as staphylococcus aureus.

Parasitic infections include, but are not limited to, worm infections,in particular, intestinal worm infection.

In one embodiment, the infection is a viral infection or anintracellular bacterial infection. In a more preferred embodiment, theinfection is a viral infection by hepatitis C, hepatitis B, influenzavirus, RSV, HPV, HSV1, HSV2, and CMV.

In one embodiment, the mammal has a type of cancer. In some instances,the cancer is a type of skin cancer where the level or type of immuneresponse in the effect mammal is not adequate to alleviate the cancer.Therefore, the invention can be used to increase the immune response inmammals that are in need of an enhancement of their immune response.Thus, treatment of the cancer (e.g., skin cancer) is accomplished byinducing an enhanced immune response using an inhibitor of CCR10 or anycomponent of the signal transduction pathway associated with CCR10 or afunctional equivalent thereof of the invention.

In some instances, an inhibitor of CCR10 or any component of the signaltransduction pathway associated with CCR10 or a functional equivalentthereof of the invention is administered in combination with animmunostimulatory protein to a patient in need thereof, resulting in animproved therapeutic outcome for the patient.

The disorder or disease can be treated by administration of an inhibitorof CCR10 or any component of the signal transduction pathway associatedwith CCR10 or a functional equivalent thereof of the inventionoptionally in combination with an antigen (e.g., vaccine) to a patientin need thereof. The present invention provides a means to increase animmune response in the patient in need thereof. In some instances, theenhancement of the immune response is specific to a desired antigen(e.g. vaccine) in the patient.

In another embodiment, the compounds of the present invention may beused in combination with existing therapeutic agents used to treat skindiseases or cancer. In some instances, the compounds of the inventionmay be used in combination these therapeutic agents to enhance theantitumor effect of the therapeutic agent.

In order to evaluate potential therapeutic efficacy of the compounds ofthe invention in combination with the antitumor therapeutics describedelsewhere herein, these combinations may be tested for antitumoractivity according to methods known in the art.

In one aspect, the present invention contemplates that the modulators ofthe invention may be used in combination with a therapeutic agent suchas an anti-tumor agent including but not limited to a chemotherapeuticagent, an anti-cell proliferation agent or any combination thereof.

The invention should not be limited to any particular chemotherapeuticagent. Rather, any chemotherapeutic agent can be linked to theantibodies of the invention. For example, any conventionalchemotherapeutic agents of the following non-limiting exemplary classesare included in the invention: alkylating agents; nitrosoureas;antimetabolites; antitumor antibiotics; plant alkyloids; taxanes;hormonal agents; and miscellaneous agents.

Alkylating agents are so named because of their ability to add alkylgroups to many electronegative groups under conditions present in cells,thereby interfering with DNA replication to prevent cancer cells fromreproducing. Most alkylating agents are cell cycle non-specific. Inspecific aspects, they stop tumor growth by cross-linking guanine basesin DNA double-helix strands. Non-limiting examples include busulfan,carboplatin, chlorambucil, cisplatin, cyclophosphamide, dacarbazine,ifosfamide, mechlorethamine hydrochloride, melphalan, procarbazine,thiotepa, and uracil mustard.

Anti-metabolites prevent incorporation of bases into DNA during thesynthesis (S) phase of the cell cycle, prohibiting normal developmentand division. Non-limiting examples of antimetabolites include drugssuch as 5-fluorouracil, 6-mercaptopurine, capecitabine, cytosinearabinoside, floxuridine, fludarabine, gemcitabine, methotrexate, andthioguanine.

There are a variety of antitumor antibiotics that generally prevent celldivision by interfering with enzymes needed for cell division or byaltering the membranes that surround cells. Included in this class arethe anthracyclines, such as doxorubicin, which act to prevent celldivision by disrupting the structure of the DNA and terminate itsfunction. These agents are cell cycle non-specific. Non-limitingexamples of antitumor antibiotics include dactinomycin, daunorubicin,doxorubicin, idarubicin, mitomycin-C, and mitoxantrone.

Plant alkaloids inhibit or stop mitosis or inhibit enzymes that preventcells from making proteins needed for cell growth. Frequently used plantalkaloids include vinblastine, vincristine, vindesine, and vinorelbine.However, the invention should not be construed as being limited solelyto these plant alkaloids.

The taxanes affect cell structures called microtubules that areimportant in cellular functions. In normal cell growth, microtubules areformed when a cell starts dividing, but once the cell stops dividing,the microtubules are disassembled or destroyed. Taxanes prohibit themicrotubules from breaking down such that the cancer cells become soclogged with microtubules that they cannot grow and divide. Non-limitingexemplary taxanes include paclitaxel and docetaxel.

Miscellaneous agents include chemotherapeutics such as bleomycin,hydroxyurea, L-asparaginase, and procarbazine that are also useful inthe invention.

An anti-cell proliferation agent can further be defined as anapoptosis-inducing agent or a cytotoxic agent. The apoptosis-inducingagent may be a granzyme, a Bcl-2 family member, cytochrome C, a caspase,or a combination thereof. Exemplary granzymes include granzyme A,granzyme B, granzyme C, granzyme D, granzyme E, granzyme F, granzyme G,granzyme H, granzyme I, granzyme J, granzyme K, granzyme L, granzyme M,granzyme N, or a combination thereof. In other specific aspects, theBcl-2 family member is, for example, Bax, Bak, Bcl-Xs, Bad, Bid, Bik,Hrk, Bok, or a combination thereof.

In additional aspects, the caspase is caspase-1, caspase-2, caspase-3,caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9,caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, or acombination thereof. In specific aspects, the cytotoxic agent is TNF-α,gelonin, Prodigiosin, a ribosome-inhibiting protein (RIP), Pseudomonasexotoxin, Clostridium difficile Toxin B, Helicobacter pylori VacA,Yersinia enterocolitica YopT, Violacein, diethylenetriaminepentaaceticacid, irofulven, Diptheria Toxin, mitogillin, ricin, botulinum toxin,cholera toxin, saporin 6, or a combination thereof.

In some embodiments, an effective amount of a compound of the inventionand a therapeutic agent is a synergistic amount. As used herein, a“synergistic combination” or a “synergistic amount” of a compound of theinvention and a therapeutic agent is a combination or amount that ismore effective in the therapeutic or prophylactic treatment of a diseasethan the incremental improvement in treatment outcome that could bepredicted or expected from a merely additive combination of (i) thetherapeutic or prophylactic benefit of the compound of the inventionwhen administered at that same dosage as a monotherapy and (ii) thetherapeutic or prophylactic benefit of the therapeutic agent whenadministered at the same dosage as a monotherapy.

Dosage and Formulation (Pharmaceutical Compositions)

Administration of the therapeutic composition in accordance with thepresent invention may be continuous or intermittent, depending, forexample, upon the recipient's physiological condition, whether thepurpose of the administration is therapeutic or prophylactic, and otherfactors known to skilled practitioners. The administration of thecompositions of the invention may be essentially continuous over apreselected period of time or may be in a series of spaced doses. Bothlocal and systemic administration is contemplated. The amountadministered will vary depending on various factors including, but notlimited to, the composition chosen, the particular disease, the weight,the physical condition, and the age of the mammal, and whetherprevention or treatment is to be achieved. Such factors can be readilydetermined by the clinician employing animal models or other testsystems which are well known to the art

One or more suitable unit dosage forms having the therapeutic agent(s)of the invention, which, as discussed below, may optionally beformulated for sustained release (for example using microencapsulation,see WO 94/07529, and U.S. Pat. No. 4,962,091 the disclosures of whichare incorporated by reference herein), can be administered by a varietyof routes including parenteral, including by intravenous andintramuscular routes, as well as by direct injection into the diseasedtissue. For example, the therapeutic agent may be directly injected intothe tumor. The formulations may, where appropriate, be convenientlypresented in discrete unit dosage forms and may be prepared by any ofthe methods well known to pharmacy. Such methods may include the step ofbringing into association the therapeutic agent with liquid carriers,solid matrices, semi-solid carriers, finely divided solid carriers orcombinations thereof, and then, if necessary, introducing or shaping theproduct into the desired delivery system.

When the therapeutic agents of the invention are prepared foradministration, they are preferably combined with a pharmaceuticallyacceptable carrier, diluent or excipient to form a pharmaceuticalformulation, or unit dosage form. The total active ingredients in suchformulations include from 0.1 to 99.9% by weight of the formulation. A“pharmaceutically acceptable” is a carrier, diluent, excipient, and/orsalt that is compatible with the other ingredients of the formulation,and not deleterious to the recipient thereof. The active ingredient foradministration may be present as a powder or as granules; as a solution,a suspension or an emulsion.

Pharmaceutical formulations containing the therapeutic agents of theinvention can be prepared by procedures known in the art using wellknown and readily available ingredients. The therapeutic agents of theinvention can also be formulated as solutions appropriate for parenteraladministration, for instance by intramuscular, subcutaneous orintravenous routes.

The pharmaceutical formulations of the therapeutic agents of theinvention can also take the form of an aqueous or anhydrous solution ordispersion, or alternatively the form of an emulsion or suspension.

Thus, the therapeutic agent may be formulated for parenteraladministration (e.g., by injection, for example, bolus injection orcontinuous infusion) and may be presented in unit dose form in ampules,pre-filled syringes, small volume infusion containers or in multi-dosecontainers with an added preservative. The active ingredients may takesuch forms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredients may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilization from solution, for constitution witha suitable vehicle, e.g., sterile, pyrogen-free water, before use.

It will be appreciated that the unit content of active ingredient oringredients contained in an individual aerosol dose of each dosage formneed not in itself constitute an effective amount for treating theparticular indication or disease since the necessary effective amountcan be reached by administration of a plurality of dosage units.Moreover, the effective amount may be achieved using less than the dosein the dosage form, either individually, or in a series ofadministrations.

The pharmaceutical formulations of the present invention may include, asoptional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, and salts of the type that arewell-known in the art. Specific non-limiting examples of the carriersand/or diluents that are useful in the pharmaceutical formulations ofthe present invention include water and physiologically acceptablebuffered saline solutions, such as phosphate buffered saline solutionspH 7.0-8.0.

The expression vectors, transduced cells, polynucleotides andpolypeptides (active ingredients) of this invention can be formulatedand administered to treat a variety of disease states by any means thatproduces contact of the active ingredient with the agent's site ofaction in the body of the organism. They can be administered by anyconventional means available for use in conjunction withpharmaceuticals, either as individual therapeutic active ingredients orin a combination of therapeutic active ingredients. They can beadministered alone, but are generally administered with a pharmaceuticalcarrier selected on the basis of the chosen route of administration andstandard pharmaceutical practice.

In general, water, suitable oil, saline, aqueous dextrose (glucose), andrelated sugar solutions and glycols such as propylene glycol orpolyethylene glycols are suitable carriers for parenteral solutions.Solutions for parenteral administration contain the active ingredient,suitable stabilizing agents and, if necessary, buffer substances.Antioxidizing agents such as sodium bisulfate, sodium sulfite orascorbic acid, either alone or combined, are suitable stabilizingagents. Also used are citric acid and its salts and sodiumEthylenediaminetetraacetic acid (EDTA). In addition, parenteralsolutions can contain preservatives such as benzalkonium chloride,methyl- or propyl-paraben and chlorobutanol. Suitable pharmaceuticalcarriers are described in Remington's Pharmaceutical Sciences, astandard reference text in this field.

The active ingredients of the invention may be formulated to besuspended in a pharmaceutically acceptable composition suitable for usein mammals and in particular, in humans. Such formulations include theuse of adjuvants such as muramyl dipeptide derivatives (MDP) or analogsthat are described in U.S. Pat. Nos. 4,082,735; 4,082,736; 4,101,536;4,185,089; 4,235,771; and 4,406,890. Other adjuvants, which are useful,include alum (Pierce Chemical Co.), lipid A, trehalose dimycolate anddimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, and IL-12.Other components may include a polyoxypropylene-polyoxyethylene blockpolymer (Pluronic®), a non-ionic surfactant, and a metabolizable oilsuch as squalene (U.S. Pat. No. 4,606,918).

Additionally, standard pharmaceutical methods can be employed to controlthe duration of action. These are well known in the art and includecontrol release preparations and can include appropriate macromolecules,for example polymers, polyesters, polyamino acids, polyvinyl,pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethylcellulose or protamine sulfate. The concentration of macromolecules aswell as the methods of incorporation can be adjusted in order to controlrelease. Additionally, the agent can be incorporated into particles ofpolymeric materials such as polyesters, polyamino acids, hydrogels,poly(lactic acid) or ethylenevinylacetate copolymers. In addition tobeing incorporated, these agents can also be used to trap the compoundin microcapsules.

Accordingly, the pharmaceutical composition of the present invention maybe delivered via various routes and to various sites in a mammal body toachieve a particular effect (see, e.g., Rosenfeld et al., 1991;Rosenfeld et al., 1991a). One skilled in the art will recognize thatalthough more than one route can be used for administration, aparticular route can provide a more immediate and more effectivereaction than another route. Local or systemic delivery can beaccomplished by administration comprising application or instillation ofthe formulation into body cavities, inhalation or insufflation of anaerosol, or by parenteral introduction, comprising intramuscular,intravenous, peritoneal, subcutaneous, intradermal, as well as topicaladministration.

The active ingredients of the present invention can be provided in unitdosage form wherein each dosage unit, e.g., a teaspoonful, tablet,solution, or suppository, contains a predetermined amount of thecomposition, alone or in appropriate combination with other activeagents. The term “unit dosage form” as used herein refers to physicallydiscrete units suitable as unitary dosages for human and mammalsubjects, each unit containing a predetermined quantity of thecompositions of the present invention, alone or in combination withother active agents, calculated in an amount sufficient to produce thedesired effect, in association with a pharmaceutically acceptablediluent, carrier, or vehicle, where appropriate. The specifications forthe unit dosage forms of the present invention depend on the particulareffect to be achieved and the particular pharmacodynamics associatedwith the pharmaceutical composition in the particular host.

These methods described herein are by no means all-inclusive, andfurther methods to suit the specific application will be apparent to theordinary skilled artisan. Moreover, the effective amount of thecompositions can be further approximated through analogy to compoundsknown to exert the desired effect.

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

EXAMPLES

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

Example 1: CCR10 Regulates Balanced Maintenance and Function of ResidentRegulatory and Effector T Cells to Promote Immune Homeostasis in theSkin

The results presented herein demonstrate that that CCR10 is a criticalregulator of immune homeostasis in the skin. The results show that inCCR10-knockout/EGFP-knockin (CCR10^(−/−)) mice (Jin et al., 2010, JImmunol 185:5723-31; Hu et al., 2011, Proc Natl Acad Sci USA108:E1035-44), imbalanced presence and dysregulated functions ofmemory-like resident regulatory and effector T cells (Treg and Teff)result in enhanced/prolonged innate and memory immune responses to theskin stimulation. CCR10 expression on the memory-like resident T cellsis preferentially imprinted early on their progenitors during skininflammation for their maintenance in homeostatic skin after resolutionof inflammation but not on skin-infiltrating effector T cells for theirmigration during inflammation. Chronic inflammation due to absence ofFoxp3⁺ regulatory T cells prevents establishment of CCR10⁺ residenteffector T cells in the skin. In addition, the enhanced immune responsedue to CCR10-knockout helps better clearance of infection of Leishmamiaparasites in the skin, suggesting clinical potentials of targeting theCCR10/ligand axis.

The materials and methods employed in the experiments disclosed hereinare now described.

Material and Methods

Mouse Models and Human Bio-Samples

CCR10-knockout/EGFP-knockin (CCR10^(−/−)) mice were generated in thelaboratory (Jin, Y., et al., 2010, J Immunol 185:5723-5731). Rag1^(−/−),Scurfy and wild type (WT) CD45.1⁺ congenic C57BL6 mice were from TheJackson Laboratory (Bar Harbor, Me.). CD45.1⁺CD45.2⁺ wild type C57BL6,CD45.1⁺CD45.2⁺ or CD45.1⁺CD45.2⁻ CCR10^(+/−), CD45.1⁺CD45.2⁺ Rag1^(−/−)mice were generated by proper crossing. Scurfy mice were also crossed toCCR10-knockout/EGFP-knockin mice to introduce aCCR10-knockout/EGFP-knockin allele for the EGFP reporter of CCR10expression. All animal experiments were approved by The PennsylvaniaState University Institutional Animal Care and Use Committee. The humanhealthy skin was from people undergoing the plastic surgery. Use of thebio-samples of humans was approved by the institutional review board ofAnhui Medical University.

Chemical Reagents and Induction of Skin Inflammation

1-Fluoro-2,4-dinitrobenzene (DNFB), Phorbol 12-myristate 13-acetate(TPA) and Fluorescein 5(6)-isothiocyanate (Fitc) and chicken ovalbumin(OVA) were purchased from Sigma-Aldrich (St. Louis, Mo.). Cholera toxinwas purchased from List Biological (Campbell, Calif.).

To induce classic contact hypersensitive (CHS) responses, mouse abdomenwas shaved and sensitized with 100 μl 0.5% DNFB in 4:1 acetone/olive oilat day 0 and 1. At day 5, the baseline ear thicknesses of both right andleft ears were measured by a micrometer gauge. Immediately following theear measurement, each side of the ear was topically applied with 10 μlof 0.2% DNFB solution or control solvents (20 μl total). Ear thicknesswas measured at various days after the chemical challenge on the ear.The change in the ear thickness (ΔT) was calculated by subtracting theear thickness before the chemical treatment from the ear thickness afterthe chemical application.

The memory CHS response was induced similarly as the classic CHSresponse except that ears were challenged with DNFB one month after theDNFB sensitization.

For DNFB, FITC or TPA-induced innate skin inflammation, each side of aear was applied with 10 μl of the chemicals (0.5% DNFB in 4:1acetone/olive oil, 0.5% FITC in 1:1 acetone/dibutylpthalate, or 100μg/ml TPA in acetone) once. The ear thickness was measured at variousdays after the application.

The OVA-induced skin inflammation was performed as reported (Campbell,J. J., et al., 2007, J Immunol 178:3358-3362), except that total OVAproteins instead of peptides were epicutaneously applied to the mouseskin.

Skin Cell Isolation

Skin cells were prepared similarly as previous described (Jin, Y., etal., 2010, J Immunol 185:5723-5731). Briefly, mouse hair was removedfrom the skin by hair clipper and Nair (Church & Dwight, Princeton,N.J.). Mouse skin was excised, trimmed of subcutaneous fat and minced,following by 2-hour digestion with 4 mg/ml Collagenase Type I(Worthington, Lakewood, N.J.), 2 mg/ml Collagenase Type IV (Worthington,Lakewood, N.J.), 2 mg/ml hyaluronidase type I-s (Sigma-Aldrich, St.Louis, Mo.) and 4% BSA (Sigma-Aldrich, St. Louis, Mo.) in DMEM. Thirtyminutes before the end of digestion, 0.0001% DNase (Sigma-Aldrich, St.Louis, Mo.) was added into the digest buffer. Mononucleocytes wereenriched from the cell preparations using Percoll gradients (40%/80%).The similarly isolated human skin cells were let recovered in theculture medium overnight before flow cytometric analysis.

Bone Marrow Cell Reconstitution

Cell sorter-purified EGFP⁻ BM cells of CCR10^(+/−) (CD45.1⁺CD45.2⁻) andCCD10^(−/−) (CD45.1⁻ CD45.2⁺) mice were 1:1 mixed and injectedintravenously into lethally irradiated (950 Rad) WT C57BL6 orCCR10^(+/−) (CD45.1⁺D45.2⁺) mice (total 10⁶ cells per mouse). Therecipients were analyzed 7 to 8 wk after the transfer.

Skin Leishmania major Infection

L. major NIH Friedlin V1 strain (MHOM/IL/80/FN) was grown in M199 mediumsupplemented with 25 mM HEPES and 20% FCS until a stationary phase. Micewere injected subcutaneously with 1×10⁶ stationary-phase promastigotesin the ear dermis. Infected ears were collected at various time pointsand total genomic DNA was extracted with DNeasy Tissue Kit (Qiagen,Valencia, Calif.). Tissue Leishmania genomic DNA levels were quantifiedby qPCR with primers (JW11: CCTATTTTACACCAAC CCCCAGT (SEQ ID NO: 1);JW12: GGGTAGGGGCGTTCTGC GAAA (SEQ ID NO: 2)) specific to L. major 16SrDNA and normalized on levels of mouse genomic β-actin as previouslydescribed (Nicolas, L., et al., 2000, Infect Immun 68:6561-6566).

In Vivo T Cell Transfer

EGFP⁻ CD3⁺ T cells were sorter-purified from splenocytes of CCR10^(+/−)or CCR10^(−/−) mice. 4×10⁵ splenic EGFP⁻ CD3⁺ T cells of CCR10^(+/−) orCCR10^(−/−) mice were intraperitoneally injected into Rag1^(−/−) miceseparately, which were then tested for the DNFB-induced classic ormemory CHS responses after the cell transfer. For adoptive transfer ofOVA-specific T cells, about 0.5×10⁶ purified splenic transgenic T cellsof CCR10^(+/−) or CCR10^(−/−) OTI or OT-II mice were intraperitoneallyinjected into WT mice with different CD45 polymorphism (indicated in therelevant figure), which were then epicutaneously challenged with Ovaproteins (Campbell, J. J., et al., 2007, J Immunol 178:3358-3362).

NaïVe T Cell Transfer-Induced Skin Inflammation

EGFP⁻ naive T cells (CD3⁺CD4⁺CD25−CD45RB^(high), CD45.1⁺) and Treg cells(CD3⁺CD4⁺CD25⁺CD45RB⁻, CD45.2⁺) were sorted from splenocytes ofCCR10^(+/−) mice. 4×10⁵ naive T cells alone, or 2×10⁵ naive along with2×10⁵ Treg cells, were intravenously injected into Rag1^(−/−) mice(CD45.1⁺CD45.2⁺). The recipients were analyzed 7 to 8 wk after thetransfer.

Real-Time RT-PCR Analysis of Cytokine Transcripts

Total RNA of mouse ears were isolated and reverse-transcribed to cDNA,which were then subject to the Sybr green real-time PCR with followingprimers. TNF-α Forward: TTCTATGGCCCAGACCC (SEQ ID NO: 3), Reverse:GGCACCACTAGTTGGTTGTC (SEQ ID NO:4); IL-1β: Forward: TCTCGCAGCAGCACATCA(SEQ ID NO: 5), Reverse: CACACCAGCAGGTTATCATCAT (SEQ ID NO: 6); IL-10Forward: ACCAAAGCCACAAAGCAGCC (SEQ ID NO: 7), Reverse:CCGACTGGGAAGTGGGTGC (SEQ ID NO: 8); β-actin Forward: CCCATCTACGAGGGCTAT(SEQ ID NO: 9), Reverse: TGTCACGCACGATTTCC (SEQ ID NO: 10). Relativelevels of transcripts were calculated with the delta delta CT method.

Antibodies and Flow Cytometry

The antibodies used for the flow cytometry and staining and analyzingstrategies were detailed in the supplementary methods. Cells stainedwith proper combination of antibodies were analyzed on a flow cytometerwith appropriate cell gating (indicated in the figures).

PE- or PeCy7-anti-mouse CD62L (MEL-14, 1:200 dilution), PE-, PeCy5- orPeCy7-anti-mouse CD44 (IM7, 1:400), PE-, Biotin- or PeCy7-anti-mouse CD4(GK1.5, 1:200), PE-, PeCy5- or Biotin-anti-mouse CD8α (53-6.7, 1:100),PE- or Biotin-anti-mouse TCRγδ (GL3, 1:100), Alexa Fluor647-anti-mouse/rat Foxp3 (FJK-16s, 1:50) and PE-anti-mouse TCRβ(H57-597, 1:100) were purchased from eBioscience (San Diego, Calif.).Alexa Fluor 647-anti-mouse IL-17A (TC11-18H10.1, 1:250), PeCy7- orPacific Blue anti-mouse CD45.1 (A20, 1:200), Alexa Fluor 647- or AlexaFluor 700-anti-mouse CD45.2 (104, 1:100), Alexa Fluor 647-anti-mouseCD127 (A7R34, 1:100), PE- or PeCy7- or Biotin-anti-mouse CD3ε (145-2C11,1:100), PE-CD25 (PC61, 1:100) and PE-KLRG1 (2F1/KLRG1, 1:100) were fromBiolegend (San Diego, Calif.). APC-anti-mouse IL-10 (JES5-16E3, 1:100),FITC-anti-human CD3 (UCHT1, 1:50), PeCy7-anti-human CD4 (SK3, 1:50) andStreptavidin-PE-Texas Red (1:200) were from BD Biosciences (San Jose,Calif.). PE-anti-human CCR10 (314305, 1:10) was from R&D Systems(Minneapolis, Minn.). PE-Alexa Fluor 610-anti-mouse CD4 (RM4-5, 1:100)and PE-Alexa Fluor 610-anti-mouse CD8α (5H10, 1:100) were fromInvitrogen (Camarillo, Calif.). Foxp3/Transcription Factor StainingBuffer Set was from eBioscience (San Diego, Calif.). Cells stained withproper combination of antibodies and analyzed on flow cytometer FC500(Beckman Coulter) or BD Fortessa LSRII (San Jose, Calif.). Cells stainedwith a single individual antibody were used to calibrate the signaloutput and compensation. Staining with the isotype control antibodieswas included to confirm the specific signal for their correspondingantigen-specific antibody staining Flow data were analyzed with Flowjo(Ashland, Oreg.).

In Vitro T Cell Stimulation

Skin EGFP⁺CD4⁺CD25⁻ T cells purified from CCR^(+/−) and CCR10^(−/−) micewere cultured in the presence of IL-2 and coated anti-CD28/anti-TCRβantibodies for 2 days. The culture media were collected and analyzed bythe mouse IL-17A ELISA Ready-SET-Go!® Set (eBioscience, San Diego,Calif.).

Statistical Analyses

Data are expressed as means±standard errors (SEM) and analyzed bytwo-tailed student T test or Fisher test to determine statisticalsignificance for two-group comparison. The ANOVA test with Tukeyadjustment was used for multiple group comparison. P<0.05 is consideredsignificant.

The results of the experiments presented in this Example are nowdescribed.

Over-Reactive Innate Immune Response to Allergen Stimulation in Skin ofCCR10^(−/−) Mice

To assess roles of CCR10 in the skin inflammation, experiments wereperformed to test CCR10−/− mice in several models. In a DNFB-induced CHSassay, CCR10^(−/−) and CCR10^(+/−) mice had similar ear thicknessincreases (FIG. 1A), indicating that CCR10 is not critical for theT-cell mediated CHS response and consistent with the report thatCCR10-knockout does not affect the T cell migration (Tubo, N. J., etal., 2011, Am J Pathol 178:2496-2503). Surprisingly, CCR10^(−/−) micehad significantly larger ear thickness increases than CCR10^(+/−) miceearly after the one-time DNFB application (FIG. 1B), suggesting anenhanced innate response. Supporting this, the treated ears ofCCR10^(−/−) mice exhibited much higher levels of gene expression ofTNF-α and lower IL-10 than CCR10^(+/−) controls (FIG. 1C). CCR10^(−/−)mice also had an enhanced innate response to one-time treatment ofanother irritant/allergen FITC (FIG. 1D-1E). On the other hand,CCR10^(−/−) and CCR10^(+/−) mice had similar responses to topicalapplication of TPA, a strong mitogen that activates both keratinocytesand immune cells (FIG. 1F and FIG. 8). Considering the one-time DNFB orFITC treatments are weaker stimulators than TPA or repeated DNFBtreatments of the CHS assay, these results reveal that CCR10^(−/−) micehave a reduced activation threshold to the weak stimulation but stillmount full-scale responses once the threshold is overcome by strongstimulators, suggesting a defect in immune regulatory system in theskin.

Imbalanced Maintenance of Skin-Resident Treg and Teff Cells inCCR10^(−/−) Mice

The next experiment was performed to analyze how CCR10-knockout affectedthe skin-resident T cells, including Treg cells that are critical in theimmune regulation. Based on the EGFP reporter for CCR10 in CCR10^(+/−)mice (Jin, Y., et al., 2010, J Immunol 185:5723-5731 and Hu, S., et al.,2011, Proc Natl Acad Sci USA 108:E1035-1044), the majority ofskin-resident αβT cells highly expressed CCR10 (EGFP) (FIG. 2A and FIG.9) and memory T cell markers (KLRG1⁻CD62L⁻ CD44^(high)CD127⁺ for CD8⁺and KLRG1^(±)CD62L⁻CD44^(high)CD127⁺ for CD4⁺ cells) (FIG. 10) (Jin, Y.,et al., 2010, J Immunol 185:5723-5731 and Kalia, V., et al., 2010,Immunity 32:91-103). Compared to CCR10^(+/−) mice, CCR10^(−/−) mice hadfewer skin αβT (CD3⁺γδTCR⁻) cells (FIG. 2A), of which CD8⁺ subset wasimpaired more profoundly than CD4⁺ subset (FIG. 2B-2C). The CCR10^(low)CD3^(high) population of T cells were Vγ3⁺ γδT cells that were increasedin the dermis of CCR10^(−/−) mice due to dysregulated localization (FIG.2A), as previously reported (Jin, Y., et al., 2010, J Immunol185:5723-5731). Notably, CCR10^(−/−) mice had significantly fewer Foxp3⁺Treg cells within the skin CD4⁺ population than CCR10^(+/−) mice (FIG.2D-2E). When total cell yields are considered, numbers of Treg, CD4⁺ andCD8⁺(CD4⁻) Teff cells isolated from skin of CCR10^(−/−) mice were allless than those of corresponding CCR10^(+/−) cells (FIG. 2F). Incontrast to the skin, very few EGFP⁺ T cells were found in internallymphoid organs such as spleens and their numbers were even higher inCCR10^(−/−) than in CCR10^(+/−) mice (FIG. 11), suggesting thatdefective maintenance of T cells in the skin results in their abnormalaccumulation in the internal sites.

Impaired Immune Homeostasis in the Skin of CCR10^(−/−) Mice

The imbalanced presence of resident Treg vs. Teff cells could impairskin immune homeostasis in CCR10^(−/−) mice. Indeed, skin CD4⁺ T cellsof CCR10^(−/−) mice contained significantly lower percentages of IL-10⁺and higher IL-17A⁺ (Th17) cells than CCR10^(+/−) controls (FIG. 2G-2H).EGFP⁺ Th17 cells of CCR10^(−/−) mice also had markedly higher meanfluorescent intensity (MFI) for the IL-17A staining than EGFP⁺ Th17cells of CCR10^(+/−) mice (FIG. 2H), suggesting that CCR10 regulatesfunction of the resident CCR10⁺ Th17 cells besides their maintenance.Supporting this conclusion, purified EGFP⁺ skin CD4⁺ Teff cells ofCCR10^(−/−) mice secreted much higher levels of IL-17A than theCCR10^(+/−) controls in culture (FIG. 21). Notably, EGFP (CCR10)⁻ skin Tcells of CCR10^(+/−) mice contained higher percentage of Th17 cells withhigher IL-17A production than EGFP (CCR10)⁺ skin T cells of the samemice (FIG. 2H), also supporting the role of CCR10 in suppression of Th17functions.

To determine whether CCR10 is directly involved in regulating the skinTreg and Teff subsets, competitive bone marrow (BM) co-transferexperiments were performed in which similar numbers of CCR10^(−/−) andCCR10^(+/−) BM cells were injected into irradiated WT mice. EGFP⁺ CD8⁺,CD4⁺ and Foxp3⁺ T cell subsets of the CCR10^(−/−) donor were all greatlyunder-represented in the skin compared to corresponding populations ofthe CCR10^(+/−) donor (FIG. 2J, FIG. 12A). In addition, EGFP⁻ CD8⁺, CD4⁺and Treg cell subsets of the CCR10^(−/−) donor were alsounder-represented (to lesser extent than the EGFP⁺ subsets) in the skincompared to corresponding populations of the CCR10^(+/−) donor (FIG.12B). Since EGFP⁻ cells do not express CCR10, the effect of CCR10-KO onthese cells is likely secondary to the effect of CCR10-KO on EGFP⁺cells. Furthermore, EGFP⁺ skin CD4⁺ T cells of the CCR10^(−/−) donorstill had higher percentages of Th17 cells with higher IL17 MFI stainingthan those of the CCR10^(+/−) donor (FIG. 2K), demonstrating anintrinsic regulatory role of CCR10 in maintenance and function of theskin-resident T cells.

Treg Cells-Regulated Immune Homeostasis is Critical for Maintenance ofCCR10⁺ Memory-Like Resident T Cells in the Skin

While the imbalanced presence and dysregulated function of skin-residentTreg vs. Teff cells are consistent with the over-reactive innateresponse in CCR10^(−/−) mice, the normal CHS response (FIG. 1A) suggeststhat CCR10 is not critical for migration of infiltrating effector Tcells during the inflammation. It was noted that DNFB-inflamed skin ofthe CHS assay had significantly lower percentages of CCR10⁺ T cells thanuntreated skin (FIG. 3A), which suggest that CCR10 is not expressed onmany new infiltrating T cells and might explain the different effects ofCCR10-knockout on skin-resident T cells under steady conditions vs.infiltrating T cells during inflammation. Dissecting this further, EGFP⁻splenic T cells of CCR10^(+/−) or CCR10^(−/−) mice were transferred intoRag1^(−/−) mice and analyzed the skin-infiltrating donor T cellsdifferent time after induction of CHS. At the peak of inflammation (1day after the DNFB challenge), only few skin-infiltrating T cells wereEGFP⁺ (FIG. 3B). In marked contrast, 1 month after the DNFB challengewhen inflammation was resolved, about half of skin T cells ofCCR10^(+/−) donor were EGFP⁺, and the percentage of EGFP⁺ skin T cellsof CCR10^(−/−) donor was significantly lower than that of CCR10^(+/−)donor (FIG. 3C-3D). Since the skin EGFP⁺ T cells of the donor inRag1^(−/−) recipients had preferentially displayed memory phenotypesfrom early on even at the active phase of inflammation (FIG. 3E), theseresults suggest that CCR10 is preferentially imprinted on memory T cellprogenitors for their maintenance in the skin after resolution ofinflammation but not on effector T cells for their skin infiltrationduring inflammation.

Experiments were designed to test this notion further using two chronicinflammation models. First, Treg-depleted splenic naïve CD4⁺ T cellswere transferred into Rag1^(−/−) mice, which induces the inflammation inthe skin (and other tissues) due to absence of Treg cells (Hong, K., etal., 1999, J Immunol 162:7480-7491 and Ma, H. L., et al., 2008, J ClinInvest 118:597-607). Nearly no skin T cells of the donor expressed EGFPin the recipients two months after the transfer (FIG. 3F, right), whilein Rag1^(−/−) mice receiving splenic naïve CD4⁺ T and Treg cells, highpercentages of skin T cells of both donor origins were EGFP⁺ (FIG. 3F,left), indicating that Treg-regulated immune homeostasis is critical forestablishment and maintenance of CCR10⁺ T cells in skin. Consistent withthis, only a few skin T cells were EGFP⁺ in Foxp3-deficient Scurfy mice,which lack Treg cells and develop the skin inflammation soon after thebirth (FIG. 3G). Together, these results reveal an important role ofCCR10 in a positive feedback circuit of immune homeostatic regulation inthe skin where CCR10 maintains balanced presence and function ofresident Treg vs. Teff cells, which in turn help to establish ahomeostatic environment important for maintenance of the CCR10⁺ residentT cells.

CCR10 is Critical for Localization of CCR10⁺ T Cells into theHomeostatic Skin

A drawback with the DNFB/FITC model is difficulty to followantigen-specific T cells. Therefore, CCR10^(−/−) mice were crossed toOT-I or OT-II mice that carry CD8⁺ or CD4⁺ T cells expressing transgenicαβTCRs specific for OVA antigens respectively. Purified naïve splenicEGFP⁻ CCR10^(+/−) and CCR10^(−/−) OT-I (or OT-II) T cells weretransferred into WT mice, followed by epicutaneous immunization with OVAon the ear (twice with a week interval) (Campbell, J. J., et al., 2007,J Immunol 178:3358-3362). One week after the second immunization, theinflamed ear and un-inflamed torso skin were analyzed for theOVA-specific T cells.

In the inflamed ear skin, the newly infiltrating OVA-specific CD8⁺ OT-IT cells accounted for the majority of total T cells (FIG. 4A). However,only a small portion of them was EGFP⁺ and CCR10-KO did not affectpercentages of the EGFP⁺ OT-I T cells in the ear (FIGS. 4A and 4C). Instriking contrast, much higher percentages of infiltrating OT-I T cellsin the untreated (and un-inflamed) torso skin were EGFP⁺ and CCR10-KOsignificantly reduced the EGFP⁺ OT-I cells in the untreated skin (FIGS.4B and 4C), revealing that CCR10 is critical for localization of theCCR10⁺ CD8⁺ T cells into un-inflamed but not inflamed skin even in thesame mouse. Similarly, CCR10 is also required for localization of CCR10⁺CD4⁺ OT-II T cells into the untreated skin but dispensable for theirlocalization into inflamed skin stimulated by the Ova immunization (FIG.13).

Defective Resolution of Immune Memory Responses in Skin of CCR10^(−/−)Mice

Skin-resident memory T cells play important roles in memory responses(Clark, R. A., 2010, J Invest Dermatol 130:362-370, Gebhardt, T., etal., 2009, Nat Immunol 10:524-530, Jiang, X., et al., 2012, Nature483:227-231 and Mackay, L. K., et al., 2012, Proc Natl Acad Sci USA109:7037-7042). CCR10^(−/−) mice were tested using a memory CHS assay,in which mice were challenged with DNFB one month (instead of five daysin a classic CHS assay) after the initial DNFB sensitization. Comparedto CCR10^(+/−) mice, CCR10^(−/−) mice had strikingly prolongedinflammation in the skin, associated with enhanced TNF-α and IL-1β andreduced IL-10 expression (FIG. 5A-5C), suggesting that the imbalancedmaintenance and function of the resident Treg vs. Teff cells also causesdefective resolution of memory responses. Further supporting thisnotion, memory CHS responses in Rag1^(−/−) mice transferred with splenicCCR10^(−/−) T cells, which had defective maintenance of thedonor-derived EGFP⁺ memory-like T cells in the skin after resolution ofinflammation in classic CHS (FIG. 3C-3D), were also prolonged comparedto the recipients of CCR10^(+/−) T cells (FIG. 5D). Notably, while therewas no difference in the early memory response between immunizedCCR10^(+/−) and CCR10^(−/−) mice the first day after re-application ofDNFB (FIG. 5A), Rag1^(−/−) mice receiving CCR10^(−/−) T cells had slowermemory responses than Rag1^(−/−) mice receiving CCR10^(+/−) T cells(FIG. 5D, Day 1). The different effects of CCR10-KO on the earlyresponse between the two models are likely because in direct comparisonof CCR10^(−/−) and CCR10^(+/−) mice, the memory response could beaffected by CCR10-regulated αβT as well as other cells while inRag1^(−/−) mice transferred with splenic T cells, some of theCCR10-regulated cells such as the epidermis-resident γδT cells are notreconstituted (Jin, Y., et al., 2010, J Immunol 185:5723-5731). Theepidermis-resident γδT cells are known to impact the skin immuneresponse (Girardi, M., et al., 2002, J Exp Med 195:855-867).

Enhanced Immune Response to and Accelerated Clearance of Leishmaniamajor Infection in the Skin of CCR10^(−/−) Mice

The finding of the important role of CCR10 in regulating theskin-resident Treg and Teff cells to prevent over-active immuneresponses predicts that targeting CCR10 might enhance immune responsesagainst skin-specific infections and other diseases. As a test,CCR10^(−/−) mice was infected with Leishmania major, a parasiticpathogen that could manipulate Treg cells to evade immune attack for itslong-term survival in skin (Belkaid, Y., et al., 2002, Nature420:502-507). Indeed, CCR10^(−/−) mice cleared the parasite much fasterthan CCR10^(+/−) mice (FIG. 6A). Consistent with an enhanced immuneresponse, CCR10^(−/−) mice had higher TNF-α and IL-1β production thanCCR10^(+/−) mice early post the infection (FIG. 6B), as in the case ofchemical challenge (FIG. 1). In addition, 1 month post the infection,significantly higher percentages of CCR10^(−/−) mice developed visibleinflammation at infection sites than CCR10^(+/−) mice did [76% (13/17)vs. 38% (8/21), P=0.025](FIG. 6C), and the infected ears of CCR10^(−/−)mice had many more T cells, most of which were EGFP⁻, than theCCR10^(+/−) controls (FIG. 6D; FIG. 14), demonstrating that absence ofCCR10 increased effector T cell response for more efficient clearance ofthe infection in the skin.

Significant Percentages of T Cells in Healthy Human Skin Express CCR10

In humans, all blood CCR10⁺ T cells display memory cell markers(Morales, J., et al., 1999, Proc Natl Acad Sci USA 96:14470-14475 andSoler, D., et al., 2003, Blood 101:1677-1682). Therefore, CCR10/CCL27might promote preferential maintenance of the CCR10⁺ memory-like T cellsin the healthy skin. However, direct evidence of the enrichment ofCCR10⁺ T cells in the skin is lacking Therefore, the T cells isolatedfrom human healthy skin were analyzed for their expression of CCR10.Compared to T cells of blood, significantly higher percentages of Tcells isolated from the normal human skin expressed CCR10 (FIG. 7),correlating with the different expression of CCR10 on circulating andskin-resident T cells in mice (FIG. 2 and FIG. 11). The similarexpression pattern of CCR10 in the skin T cells of mice and humanssuggests that CCR10 might function in the similar fashion in both humanand mouse skin.

CCR10 Regulates Balanced Maintenance and Function of Resident Regulatoryand Effector T Cells to Promote Immune Homeostasis in Skin

CCR10/CCL27 are implicated in various skin allergy and inflammatorydiseases but the in vivo function of the pair has been elusive,hindering the effort in understanding their involvement in the diseasedevelopment (Homey, B., et al., 2002, Nat Med 8:157-165). In thisreport, definite evidence that CCR10 is critical in the regulation ofimmune homeostasis and resolution of inflammation is provided, which isan advance from the current focus on the role of CCR10 as a homingmolecule for infiltrating T cells to promote the immune response in theskin. Mechanistically, CCR10 could potentially function in severalaspects to regulate the skin T cells (illustrated in FIG. 15). First,CCR10 is critical in migration of CCR10⁺ memory (or memory-like) T cellprecursors into the skin under homeostatic conditions while it isdispensable for infiltration of T cells into the inflamed skin. Inaddition, CCR10 expressed on the skin-resident Treg and Teff cells isimportant for their balanced maintenance in the skin under homeostaticconditions, potentially by regulating their retention, proliferation andsurvival in the skin. Furthermore, the results presented herein alsosuggest that CCR10 might signal to regulate functions of the CCR10⁺skin-resident T cells such as their production of inflammatory andregulatory cytokines. In absence of CCR10, the dysreguated maintenanceand functions of resident Teff and Treg cells in the skin could resultin the over-active immune response and impaired resolution of skininflammation. In light of the finding of CCR10 as a critical regulatorof skin immune homeostasis in mice, roles of the CCR10/CCL27 axis in theskin allergy and inflammatory diseases in patients need to be carefullyinvestigated before targeting the axis for treatment of the diseases. Itis possible that the upregulated expression of CCL27 in the inflamedskin is secondary to the inflammation for a purpose of inflammationregulation. Therefore, developing a strategy to increase the number andactivity of the CCR10+ resident regulatory T cells might help to restorethe immune homeostasis and treat inflammatory diseases in the skin.

Considering both resident Treg and Teff cells are reduced in the skin ofCCR10-knockout mice, the over-active immune response to the skinstimulation is likely due to impaired presence and dysregulated functionof both populations. Consistent with this, the results presented hereinsuggests that CCR10 has an intrinsic regulatory role in controllingfunction of Teff cells (Th17), which is in addition to potentialregulatory effect of Treg cells on these effector T cells. Althoughmechanisms underlying the intrinsic regulatory functions of CCR10 in theTeff and Treg cells are not clear, the results presented herein isreminiscent of functions of some other T cell regulatory receptors suchas PD-1 and CTLA-4 that inhibit Teff cell functions and enhances Tregcell functions (Francisco, L. M., et al., 2009, J Exp Med 206:3015-3029and Krummel, M. F., et al., 1995, J. Exp. Med. 182:459-465). Consideringthe importance of balanced production of inflammatory and regulatorycytokines in immune homeostasis and inflammation, it would be importantto figure out the mechanism of CCR10 in regulating T cell activation andinflammatory cytokine production in the future. In addition, addressingthe origin of CCR10⁺ memory (or memory-like) Teff and Treg cells isanother important question to understand their functions.

CCR10-knockout affects the presence of CD8+ Teff and Treg subsets in theskin to a larger extent than CD4+ Teff cells, suggesting that thedifferent resident T cell subsets are intrinsically different in theirrequirements of CCR10 for maintenance. While the underlying mechanism isnot clear, it seems to be associated with the different mobility andexpression of skin retention molecules of the different T cells subsets(Sather, B. D., et al., 2007, J Exp Med 204:1335-1347, Gebhardt, T., etal., 2011, Nature 477:216-219 and Sharma, R., et al., 2009, J Immunol183:1065-1073). Of note, the skin-resident CD8⁺ memory T cells do notmove while the CD4⁺ cells are highly mobile (Sather, B. D., et al.,2007, J Exp Med 204:1335-1347, Gebhardt, T., et al., 2011, Nature477:216-219 and Sharma, R., et al., 2009, J Immunol 183:1065-1073).While mobility of skin-resident Treg cells is well studied, they expressthe skin adhesion molecules CD103 as the resident CD8⁺ cells do whilethe resident CD4⁺ Teff cells do not (Sather, B. D., et al., 2007, J ExpMed 204:1335-1347, Gebhardt, T., et al., 2011, Nature 477:216-219 andSharma, R., et al., 2009, J Immunol 183:1065-1073).

While a predominantly net effect of CCR10-knockout on the skin immuneresponse is the over-reactive and prolonged inflammation, CCR10 might beimportant for an efficient effector memory T cell response. Rag1^(−/−)mice reconstituted with CCR10^(−/−) T cells had a slower and prolongedmemory response compared to those reconstituted with CCR10^(+/−) Tcells, suggesting that both effector and resolution phases of the memoryresponse are impaired. Likely, the CCR10-dependent efficient andbalanced maintenance of resident Teff and Treg memory T cells in theskin allows a rapid effector T response to a challenge as well as properresolution of the effector response after clearance of the challenge.Such efficient effector and regulatory responses are important forpreservation of the function of skin while dealing with harmfulchallenges of environments. Dysregulation of either phase of theresponse could result in skin diseases.

Example 2: CCR10-Regulated ILC in Prevention of Skin Inflammation

The set of experiments were designed to evaluate the role of CCR10 inpreventing skin inflammation.

ILC Cells

ILC cells are a family of newly identified innate lymphocytespreferentially enriched in barrier tissues such as the intestine andskin and involved in the local tissue homeostasis and inflammation. ILCsdo not express cell-surface markers associated with other immune celllineages (lineage marker-negative or LIN−) but express the commonhematopoietic lineage marker CD45 and surface molecules commonlyassociated with lymphocytes such as CD90 and CD127. Based on theirdevelopmental pathways and functional potentials in analog to thedifferent T helper cell (Th) subsets, ILCs were divided into three majorgroups (ILC1-3). ILC1 comprises natural killer (NK) cells and other ILCsthat produce IFN-γ. ILC2 cells predominantly express Th2-type cytokinessuch as IL-5 and IL-15, and ILC3 cells produce Th-17/22 type cytokinessuch as IL-17 and IL-22. Through the production of unique cytokines anddirect cell-cell interaction, the different groups of ILCs have beenreported to act on various other subsets of immune cells, such as Tcells, mast cells, eosinophils and macrophages, and epithelial cells.

Function and Regulation of ILCs in the Skin Immune Activation andHomeostasis

ILCs were only recently found in the skin and involved in the skininflammation as well as homeostatic regulation. One early study reportedthat IL-17 producing ILCs (belonging to the ILC3 group) could infiltratethe inflammatory skin of mice in an Imiquimod (Aldara) cream-inducedpsoriasis model to contribute to the disease development. More recently,several groups reported that ILC2 cells were involved in mediating theskin inflammation in atopic dermatitis. In addition, ILC2 cells werefound to reside in the healthy skin where they interact with mast cells,implicating them in regulation of other skin-immune cells for thehomeostatic maintenance. However, mechanisms regulating theskin-specific localization of ILCs and their various functions are notclear.

CCR10 is Expressed on Most ILCs of Homeostatic Skin and Important forthe Skin Immune Homeostasis

A strain of CCR10-knockout/EGFP-knockin mice in which the CCR10 codingregion was replaced with a DNA sequence coding for enhanced greenfluorescent protein (EGFP) as a reporter for CCR10 was generated. Usingheterozygous and homozygous CCR10-knockout/EGFP-knockin (CCR10^(+/EGFP)and CCR10^(EGFP/EGFP), or CCR10^(+/−) and CCR10^(−/−) for simplicity)mice, expression of CCR10 and its roles in skin immune cells wereassessed. It was found that the CCR10+ ILCs are generated underhomeostatic conditions only in the skin-draining lymph nodes (sLN), anddependent on CCR10 as well as CCR6 for localization in the skin. TheCCR10+ ILCs were capable of producing more IL-17 as well as IL-5 thantheir CCR10− counterparts, and most of them expressed high levels ofMHCII, suggesting their role in regulation of CD4+ T cells. Supportingthis, it was found that CCR10+ sILCs were important in homeostaticmaintenance of skin regulatory T (Treg) cells to prevent effector T(Teff) cells-induced skin inflammation. Reciprocally, the generation andlocalization of CCR10+ sILCs required Treg cell-regulated homeostaticenvironments and were suppressed under inflammatory conditions,indicating a cross regulation of CCR10+ ILC and T cells in the immunehomeostatic maintenance in the skin.

CCR10+ ILCs are the Dominantly Active Population of Skin-Specific ILCsUnder Homeostatic Conditions

Based on the EGFP reporter of CCR10 expression in CCR10^(+/EGFP) (orCCR10^(+/−) for simplicity) mice, the majority of CD3⁻Lin⁻CD127⁺CD90⁺ILC cells within the CD45⁺ population of the skin expressed CCR10(EGFP⁺) but those of mucosal tissues such as intestines and lungs didnot (FIG. 16a-b ). CCR10⁺ and CCR10⁻ skin_ILCs expressed other commonepithelial tissue-homing and adhesion molecules CCR6, CD103 and CD69(FIG. 16c ), indicating that they were all resident ILCs.

CCR10⁺ and CCR10⁻ skin_ILCs also both expressed the lymphocyteactivation marker CD44 (FIG. 16d ). However, significantly higherpercentages of CCR10⁺ skin_ILCs were Ki-67⁺ than CCR10⁻ skin_ILCs (FIG.16d ), suggesting that CCR10⁺ skin_ILCs were more activating cells.Consistent with this notion, CCR10⁺ skin_ILCs had higher percentages ofIL-17⁺ (ILC3-like) and IL-5⁺ (ILC2-like) cells (FIG. 16e ). Few CCR10⁺or CCR10⁻ skin_ILCs produced IFN-γ or IL-10 (FIG. 16e ).

Previous studies with intestinal ILCs found that a fraction ofintestinal ILC3 cells expressed high levels of MHCII with regulatoryantigen-presenting cell (APC) capacities {Hepworth, 2013 #171}. Weanalyzed CCR10⁺ and CCR10⁻ skin_ILCs for their expression of MHCII andother co-stimulatory and co-inhibitory molecules. Markedly, most CCR10⁺skin_ILCs expressed high levels of MHCII while CCR10⁻ skin_ILCsexpressed no or lower levels of MHCII (FIG. 16f ). In addition, mostCCR10⁺ skin_ILCs expressed the co-inhibitory molecule PD-L1 while theyexpressed no or low levels of other co-stimulatory or co-inhibitorymolecules CD80, CD86, PD-L2, B7x, B7h, B7H3, OX40L and CD40 (FIG. 16g ),suggesting their potentials as regulatory APCs.

CCR10⁺ ILCs are Active ILCs Found in Skin-Draining Lymph Nodes but notOther Secondary Lymphoid Organs

To understand the process for the preferential establishment of CCR10⁺ILCs in the skin, we searched for CCR10⁺ ILCs in skin-draining LNs(sLNs) and other lymphoid organs. Strikingly, most ILCs of sLNs(sLN_ILCs) expressed high levels of CCR10 while ILCs of BM, spleens orintestine-draining mesenteric lymph nodes (mLNs) were CCR10⁻ (FIG. 16h-i). Similar to CCR10⁺ skin_ILCs, the vast majority of CCR10⁺ sLN_ILCsexpressed CCR6 and CD103 and most of them expressed CD69 (FIG. 16j ).

The similarity of CCR10⁺ skin_ILCs and sLN_ILCs supports the idea thatskin-specific CCR10⁺ ILCs might be selectively programmed in sLNs.Consistent with this, CCR10⁺ sLN_ILCs uniformly expressed high levels ofCD44 (FIG. 16k ), suggesting that they were activated cells. Confirmingthis, significantly high percentages of CCR10⁺ sLN_ILCs were at theproliferating phase based on their expression of Ki-67 (FIG. 16k ). Inaddition, CCR10⁺ sLN_ILCs expressed high levels of IL-17 and IL-5 but noIFN-γ or IL-10 (FIG. 16l ). Like CCR10⁺ skin_ILCs, the vast majority ofCCR10⁺ sLN_ILCs expressed high levels of MHCII (FIG. 16m ). Together,these results suggest that in sLNs, activated ILCs are predominantlyprogrammed to acquire high expression of CCR10 and other skin-homingmolecules for their migration into the skin to replenish the local ILCpool.

CCR10 and CCR6 are Critical for Localization of CCR10+ sILCs in(to) theSkin

Experiments were designed to compare CCR10^(EGFP/EGFP) andCCR10^(+/EGFP) littermates for EGFP⁺ ILCs in sLNs, skin and spleens todetermine roles of CCR10 in specific migration and establishment ofCCR10⁺ ILCs in(to) the skin. EGFP⁺ ILCs in CCR10^(EGFP/EGFP) mice are“CCR10⁺ wannabe” ILCs that do not express CCR10 proteins. Compared toCCR10^(+/EGFP) littermates, CCR10^(EGFP/EGFP) mice had similar, if nothigher, percentages and numbers of EGFP⁺MHCII⁺ ILCs in sLNs (FIG. 17A).In contrast, there was a specific decrease of EGFP⁺MHCII⁺ ILCs in theskin of CCR10^(EGFP/EGFP) mice while other subsets (EGFP⁺MHCII⁻ or EGFP⁻ILCs) increased or did not change significantly compared to theircorresponding controls in CCR10^(+/EGFP) mice (FIG. 17B). These resultssuggest that CCR10 is required for the specific localization of ILCsinto the skin but increased maintenance of other subsets of ILCs mightcompensate for the impaired migration and localization of EGFP⁺MHCII⁺ILCs in absence of CCR10. Compared to CCR10^(+/EGFP) controls, therewere significantly increased percentages of EGFP⁺ ILCs in spleens ofCCR10^(EGFP/EGFP) mice (FIG. 17C). These results suggest that in absenceof CCR10 expression, the impaired migration of EGFP⁺ ILCs into the skinis associated with their abnormal accumulation in lymphoid organs thatdo not express ligands for CCR10, indicating a critical role of CCR10 inthe skin-specific localization of CCR10⁺ ILCs.

CCR6 is expressed on the vast majority of CCR10⁺ and CCR10⁻ ILCs of sLNsand the skin (FIG. 16). Compared to CCR6-sufficient littermate controls,CCR6^(−/−) mice had no significant change in percentages of CCR10⁺MHCII⁺ILCs in the skin (FIG. 17B). Unlike CCR10^(EGFP/EGFP) mice, CCR6^(−/−)mice did not have abnormally increased accumulation of EGFP⁺ ILCs inspleens (FIG. 17C). These results suggest that CCR6-knockout itself doesnot significantly affect localization of CCR10⁺ ILCs into the skin.

Experiments were also designed to test whether CCR6 coordinated withCCR10 in regulating homeostatic presence of CCR10⁺ ILCs in the skinusing CCR10^(EGFP/EGFP)CCR6^(−/−) mice. Compared to CCR10^(+/EGFP) mice,CCR10^(EGFP/EGFP)CCR6^(−/−) mice had even fewer EGFP⁺MHCII⁺ ILCs in theskin than CCR10^(EGFP/EGFP) mice (FIG. 17B). Associated with this, therewas further increased accumulation of EGFP⁺ ILCs in spleens ofCCR10^(EGFP/EGFP)CCR6^(−/−) mice than of CCR10^(EGFP/EGFP) mice (FIG.17C). There was no significant change of EGFP⁺ or EGFP⁺MHCII⁺ ILCs insLNs of CCR10^(EGFP/EGFP)CCR6^(−/−) mice (FIG. 17A). Together, theseresults demonstrate that CCR10 and CCR6 coordinately regulate thespecific localization and maintenance of CCR10⁺ and CCR10⁻ ILCs in theskin.

Dysregulated Activation of ILCs in Skin-Draining Lymph Nodes and Absenceof CCR10⁺ ILCs in the Skin of Rag1^(−/−) Mice

Since CCR10 is also involved in localization of resident T cells in thehomeostatic skin, we crossed CCR10^(EGFP/EGFP) mice onto Rag1^(−/−)background with intention to study how CCR10⁺ ILCs were regulated andfunctioned without T cells. Surprisingly, in Rag1^(−/−)CCR10^(+/EGFP)mice, the vast majority of skin_ILCs were CCR10(EGFP)⁻ and had noexpression of MHCII (FIG. 18a ), indicating that they were differentlyactivated ILCs. The reduced CCR10⁺ skin_ILCs in Rag1^(−/−)CCR10^(+/EGFP)mice was not due to the reduced generation of CCR10⁺ ILCs in sLNs. Therewere high percentages of CCR10⁺MHCII⁺ ILCs in sLNs ofRag1^(−/−)CCR10^(+/EGFP) mice (FIG. 18b ). Interestingly, CCR10⁻ ILCs insLNs of Rag1^(−/−) CCR10^(+/EGFP) mice also displayed activatedphenotypes, as indicated by their uniform expression of high levels ofCD44 and similar percentages of Ki-67⁺ population as CCR10⁺ ILCs (FIG.18c ), which were higher than CCR10⁻ sLN_ILCs of WT mice (FIG. 18d ).These results suggest that the preferential activation/generation ofCCR10⁺ ILCs in sLNs and their homeostatic presence in the skin arecritically dependent on adaptive immune cells.

Treg Cells-Regulated Homeostatic Conditions are Critically Required forGeneration and Establishment of CCR10⁺ ILCs in Skin-Draining Lymph Nodesand the Skin

Skin-resident CD4⁺ T cells are critically involved in the skinhomeostasis. Experiments were designed to test whether transfer of CD4⁺T cells would help re-establishment of homeostatic presence of CCR10⁺ILCs in the skin of Rag1^(−/−) CCR10^(+/EGFP) mice. Indeed, adoptivetransfer of CD4⁺ splenic T cells of WT mice significantly increasedpercentages of CCR10⁺ and CCR10⁺MHCII⁺ ILCs in the skin ofRag1^(−/−)CCR10^(+/EGFP) mice (FIG. 18e ).

Treg cells are a major subset of CD4⁺ T cells critically involved inimmune homeostasis. Experiments were therefore designed to test whetherTreg cells were required for the CD4⁺ T cell-rescued CCR10⁺ ILChomeostasis in the skin. Indeed, transfer of Treg-depleted splenic CD4⁺T cells could not rescue the impaired CCR10⁺ skin_ILC homeostasis inRag1^(−/−)CCR10^(+/EGFP) mice (FIG. 18e ).

Experiments were also designed to test the requirement of Treg cells ingeneration and homeostatic presence of CCR10⁺ ILCs in sLNs and the skinusing Foxp3^(−/−) mice, which lack all Treg cells and develop the skin(and systemic) inflammation. Very few ILCs of sLNs and the skin ofFoxp3^(−/−) mice expressed CCR10 (FIG. 18f-18g ), demonstrating thatTreg-dependent homeostatic conditions were critical for the preferentialgeneration of CCR10⁺ ILCs in sLNs and their establishment in the skin.

Skin Inflammation Suppresses Generation of CCR10⁺ ILCs in Skin-DrainingLymph Nodes and their Presence in the Skin

Dysregulated activated ILCs contribute to inflammation symptoms in theskin. The specific generation and presence of CCR10⁺ ILCs in sLNs andthe skin under only homeostatic conditions suggest that they aredifferent from those mediating the skin inflammation. To further definehow inflammatory conditions affected the programming of CCR10⁺ ILCs, wetreated CCR10^(+/EGFP) mice with topical application of calcipotriol, asynthetic vitamin D3 analog that induces atopic dermatitis-like skininflammation mediated by ILC2 cells. The treatment resulted insignificant reduction of CCR10⁺ ILCs in sLNs and the skin (FIG. 18h ),suggesting that generation and establishment of CCR10⁺ ILCs in sLNs andthe skin was suppressed under these inflammatory conditions.

CCR10⁺ ILCs Regulates Homeostasis of Resident Treg Cells in the Skin

The predominance of CCR10⁺ ILCs in the healthy skin suggests that theycontribute to immune homeostatic regulation. Consistent with this idea,CCR10⁺ ILCs have molecular potentials as regulatory APCs for Th cellswith their high levels of expression of MHCII and the co-inhibitorymolecule PD-L1 but low levels of expression of co-stimulatory molecules(FIG. 16), raising the possibility that CCR10⁺ ILCs regulate homeostasisof CD4⁺ Th cells, particularly Treg cells, in the skin.

To address specifically the role of CCR10⁺ ILCs in homeostasis of Thcells in the skin, we tested whether transferred CD4⁺ splenic T cellscould be able to establish homeostasis in the skin ofCCR10^(EGFP/EGFP)Rag1^(−/−) recipient mice as efficiently as inCCR10-sufficient Rag1^(−/−) recipient mice. Compared to donor CD4⁺ Tcells in the skin of Rag1^(−/−) recipients, donor CD4⁺ T cells in theskin of CR10^(EGFP/EGFP)Rag1^(−/−) recipients had significantly reducedpercentages of Treg cells (FIG. 19a ), indicating that CCR10⁺ skin_ILCswere important for homeostatic establishment of skin Treg cells. Tofurther determine the role of ILC in homeostatic establishment ofskin-resident T cells, we transferred CD4⁺ splenic T cells intoIL2Rγ^(−/−)Rag2^(−/−) mice, which lack all ILCs. There were moreseverely reduced Treg cells of the donor origin in the skin ofIL2Rγ^(−/−)Rag2^(−/−) recipients than of CCR10^(EGFP/EGFP)Rag1^(−/−)recipients (FIG. 19b ). In addition, in contrast to donor T cells in theskin of Rag1^(−/−) or CCR10^(EGFP/EGFP)Rag1^(−/−) recipients, nearly alldonor T cells in the skin of IL2Rγ^(−/−)Rag2^(−/−) recipients wereCCR10⁻ (FIG. 19b ), indicating that the transferred CD4+ T cells couldnot establish homeostasis in the skin of IL2Rγ^(−/−)Rag2^(−/−)recipients. Consistent with this, markedly higher percentages of donor Tcells in the skin of IL2Rγ^(−/−)Rag2^(−/−) recipients expressed IL-17Athan those of Rag1^(−/−) recipients (FIG. 17c ). Together, these resultsdemonstrate that CCR10⁺ skin_ILCs are critical in homeostatic regulationof skin-resident T cells.

The results presented herein demonstrate identification of a novelpopulation of skin-resident CCR10+ ILCs that are generated in the skindLNs and involved in regulation of the CD4+ cells to promote thecutaneous immune homeostasis. Reciprocally, the generation of CCR10+ILCs in the skin dLNs and their localization/maintenance in the skin aredependent on immune homeostatic conditions regulated by T cells,particularly Tregs. These findings demonstrate that skin CCR10+ sILC andT cells inter-depend on each other to maintain the immune homeostasisand prevent the autoimmune inflammation in skin.

Example 3: CCR10 in the Prevention of Psoriasis-Like Skin Inflammation

Increased expression of IL-17 by skin immune cells of CCR10^(−/−) micesuggests that CCR10-mediated signals regulate proper expression of IL-17(FIGS. 20A and 2), an important pro-inflammatory cytokine involved inthe development of psoriasis, to suppress the disease development. Theincreased expression of IL-17 in CCR10^(−/−) skin T cells is moredrastic in old mice (FIG. 20B) while the expression of IFN-γ is furtherdecreased in old CCR10^(−/−) mice (FIG. 20B). CCR10 is also important toprevent over-expression of IL-22 since CCR10^(−/−) skin lymphocytesexpressed higher levels of IL-22 (FIG. 20A), another cytokine involvedin the psoriasis. Together, these results demonstrate that withoutCCR10, the increased expression of IL-17/IL-22 might cause increasedincidence of inflammation, especially in aged mice. Consistent with thisidea, old CCR10^(−/−) mice had enlarged spleens compared to CCR10^(+/−)control mice (FIG. 20C). To test directly whether CCR10 is important inprevention of psoriasis, we treated CCR10^(−/−) and CCR10^(+/−) micewith the topical application of imiquimod, a chemical that inducespsoriasis-like skin inflammatory symptoms. Indeed, compared toCCR10^(+/−) mice, CCR10^(−/−) mice had much more severe psoriasis-likeskin inflammation after imiquimod treatment (FIG. 20D). These resultsdemonstrate that promoting CCR10 signals could alleviate or treat thepsoriasis.

Example 4: CCR10 in the Prevention of Intestinal Inflammation

CCR10 also regulates the immune homeostasis in the mucosal sites such asthe intestines where another of its ligand, CCL28, is highly expressed.Experiments were designed to compare CCR10^(−/−) and CCR10^(+/−) mice totest the role of CCR10 as a regulator of intestinal homeostasis, whichhas important clinic relevance to inflammatory bowl diseases (IBD).

Development of Spontaneous Inflammatory Symptoms in Colons and ImpairedRecovery from DSS-Induced Colitis in CCR10^(−/−) Mice

Compared to CCR10^(+/−) mice, aging CCR10^(−/−) mice have shorter colonsassociated with higher gene expression of IL-17 and IFN-γ, indicatingspontaneous development of intestinal inflammation (FIGS. 21A and 21B).Furthermore, upon treatment with dioctyl sodium sulfosuccinate (DSS),CCR10^(−/−) mice had significantly higher frequency of fecal occultblooding than CCR10^(+/−) mice (FIG. 21C), and had increased incidenceof death (FIG. 21D). Levels of TNF-α, IL-17 and IL-22 were all higher incolons of DSS-treated CCR10^(−/−) mice than CCR10^(+/−) controls (FIG.21E). Together, these results demonstrate that CCR10 is critical in theintestinal immune homeostasis.

CCR10 is Important for the Homeostasis of Treg and Teff Cells in theIntestines to Prevent Intestinal Inflammation

The increased expression of the inflammatory cytokines such as IL-17 andIL-22 in the intestines of CCR10^(−/−) mice could be due to dysregulatedT cells. Compared to CCR10^(+/−) controls, higher percentages ofintestinal CD4+ T cells of CCR10^(−/−) mice expressed IL-17A as well asIFN-γ (FIG. 22A). In contrast, total CD4⁺ intestinal T cells had reducedpercentages of regulatory IL-10⁺ T cells due to reduced numbers of Tregcells (FIGS. 22A and 22B). Notably, EGFP+ Foxp3+ CD4+ Treg cells werereduced more significantly than EGFP+ Foxp3− CD4+ Teff cells inintestines of CCR10^(−/−) mice (FIG. 22B), suggesting that CCR10 isimportant in the balanced presence of Treg and Teff cells in theintestine. The more severe reduction of the EGFP+ intestinal Treg cells(FIG. 22B) in CCR10^(−/−) mice correlates with the finding that higherpercentages of intestinal Treg cells stain positive for EGFP (CCR10)(FIG. 22C). However, CCR10 is not directly involved in the regulation ofT cell homeostasis since intestinal T cells themselves do not expressCCR10 (FIGS. 22D and 22E). Instead, intestinal T cells form conjugateswith CCR10 (EGFP)+ IgA+ cells (FIGS. 22D and 22E), supporting a notionthat CCR10+ IgA+ cells interact with intestinal T cells to promote theirhomeostasis and prevent the intestinal inflammation. Therefore promotingthe CCR10/ligand signals could potentially reduce intestinalinflammation in IBD diseases.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed is:
 1. A method of inhibiting an immune response in amammal suffering from psoriasis, the method comprising administering tothe mammal in need thereof an effect amount of a composition comprisingan activator of CCR10, wherein the activator is CCL27.